9 Regulation of Cell cycle • Cell division • Cell division –procaryotic cells • Cell cycle • Mitosis, its individual phases • Meiosis, fundamental differences from mitosis • Control of cell division and cell growth 9-Cell cycle 2022 1 What is cell division? ▪ arranged sequence of macromolecular processes in which the cell replicates its content and then splits into two daughter cells, and each of them bears the same chromosomes ▪ main goal: the reproduction of genetic material for the next generation 9-Cell cycle 2022 2 Cell division in unicellular Unicellular: Harmonization of cell division with growth Cell division in bacteria (E.coli) It has a single circular chromosome, which is attached to the plasma membrane and remains attached to it during chromosome replication. Both chromosomes are separated from each other by cell growth. Cell wall and plasma membrane goes between chromosomes → two cells are formed. = BINARY FISSION 9-Cell cycle 2022 3 1 Origin of replication E. coli cell Two copies of origin Cell wall Plasma membrane Bacterial chromosome Origin Origin Chromosome replication begins. Replication continues. Replication finishes. Two daughter cells result. 2 3 4 Figure 12.12-4 FtsZ ring 9-Cell cycle 2022 4 Cell division -cell cycle in multicellular • at the beginning of the development of a multicellular organism is a single cell • cell division necessary for - growth of the organism - replacement of damaged and old cells - the spread of genetic information: mitosis, meiosis • before the division the cell must enlarge, chromosomes duplicated and ensure their accurate division into daughter cells • Coordination of these processes takes place within the cell cycle • cell division is strictly controlled to maintain the unity of multicellular organism • the loss of dividing control leads to abnormal development and may cause tumors In adults the cells divide only in such places and at the time when it is needed (e.g. replacement of dying cells, restoring the injured tissue) • Reproduction • Development and growth • The renewal of tissues 9-Cell cycle 2022 5 Eukaryotic cells (a) Reproduction (b) Growth and development (c) Tissue renewal 20 m 100 m 200 m Usually divide at all: neural, muscle Minimum is divided: liver cells (1 per year) Intensively divided: intestinal epithelial cells, blood precursor cells (more than 1 day). Each of us creates million new cells every second , division arrest leads to death. 9-Cell cycle 2022 6 Cell cycle cycle of cell life processes that begin with her birth and ends with the formation of daughter cells • precise continuity of cellular processes, two distinct phases: Interphase • cell growth, cell organelles duplication, increased metabolism G1 first growth phase, the active synthesis of RNA and proteins cell accumulates energy and prepares for DNA synthesis (Replication proteins, histones) S The synthesis of DNA, chromosomal duplication G2 second growth phase continues synthesis of RNA and proteins cell growth and preparation for M phase (mitotic spindle) M phase • mitosis (division of the nucleus) and cytokinesis (Cell division) • chromatin condensation • breakdown of the nuclear membrane • Creation of spindle and kinetochore • chromatid separation • breakdown of spindles • chromatin decondensation • formation of membrane, cell division 9-Cell cycle 2022 7 Phase of the cell cycle of eukaryotic cells G0 -PHASE INTERPHASE (G1, S, G2) (normal cellular activity) M-PHASE (cell division) G1 and G2 phases are phases when cell growth, duplication of cytoplasmic organelles are in progress G = gap ❖G0-phase (resting) maintained only basal metabolism ❖ It occurs only in certain cell types, especially those that have been terminally differentiated (neurons) 9-Cell cycle 2022 8 The synthesis of DNA during cell cycle all structure and functions of cells are encoded by the DNA • during cell division, each cell must obtain a complete genetic information • DNA is replicated before the cell division • there is a duplication of chromosomes • cell in G2 phase has 2x higher DNA content than in the G1 phase • resulting copies of chromosomes are joined at the centromere region, form sister chromatids which are separated during the nucleus division Synthesis of proteins and RNA during the cell cycle • still as fast during interphase • in M phase the protein synthesis decreases and the synthesis of RNA is stopped • all proteins are formed during interphase continuously • exception are the histones, which are formed only in G1 and S phase • After completion of replication, histone mRNAs are degraded 9-Cell cycle 2022 9 High accuracy requirements • correct sorting of phases • flawless replication • accurate chromosomes segregation Unwanted risks: • replication before mitosis: loss of genetic information at least in one cell • double replication of chromosomes before mitosis: increase of genes copy number 9-Cell cycle 2022 10 The length of the cell cycle is different Cell type Length of teh cell cycle Yeast cells 1,5 – 3 hours intestinal epithelium cells 12 hours mammalian fibroblasts 20 hours mammalian liver cells 1 year Time requirements of the cell cycle • the speed of the cell cycle is determined by internal and external stimuli • cells with distinct specializations without the ability to divide (neurons, muscle cells, red blood cells) • cells that divide only under certain conditions (liver cells, lymphocytes) • rapidly dividing cells (epithelial, blood stem cells) yeasts 1.5 - 3 hours, intestinal epithelium 12 hours, mammalian fibroblasts in culture for 20 hours, mammalian liver cells about 1 year interphase occupies most of the time, can last for hours, days, weeks, months, years depending on the type of cells • proliferating human cell has about 22 hour cycle • cell that stops to diferentiate enters the resting phase G0 • variability is given by length of G1 phase9-Cell cycle 2022 11 How is the accuracy of cycle achieved? ❑ the use of checkpoints, which stop the beginning of each subsequent step before completing the previous step ❑ damage of checkpoints by mutation: chromosomal rearrangements, abnormal numbers of chromosomes, changes in gene expression, etc. (cancer risk) ❑ system of regulation of DNA replication and segregation of chromosomes is in all eukaryotic cells identical ❑ similar to the transcription and translation: the management system of the cell cycle is conservative and evolutionary stable Restriction point • in late G1 phase • for transition from G1 to S an extracellular signal is needed (growth factor, mitogen) • Without this signal cell leaves the cycle and enters the G09-Cell cycle 2022 12 S-phase (synthetic) ❖ replication of nuclear DNA ❖ synthesis of histones G1-phase (presynthetic) probíhají syntetické procesy = ribozomy, ER, mitochondrie, enzymy, nukleotidy M-phase (mitotic) G2-phase (postsynthetic) ❖ protein synthesis, RNA Mitosis (karyokinesis) = division of the nucleus cytokinesis = division of the cytoplasm ❖G0-phase (resting) ❖ maintained only basal metabolism ❖ It occurs only in certain cell types, esp. those which are already terminally differentiated (neurons) The cell cycle of eukaryotic cells INTERPHASE 9-Cell cycle 2022 13 S –phase (synthetic) The cell replicates nuclear DNA - duplication of chromosomes M–phase (mitotic) Cell growth stops and is followed by 1) Nuclear division - condensation of chromosomes - formation of the mitotic spindle 2) Cytoplasmic division - formation of contractile ring - division of organelles MT and ChL are divided, GA and ER disintegrates into small fragments, which increases the probability of uniform distribution.9-Cell cycle 2022 14 G1 phase First growth phase after cell division, follows the cytokinesis • cell needs to reach its original parent cell size • in early embryos G1 and G2 phase is missing, the cell divides and diminishes creation of cytoplasm and new organelles, synthesis of RNA, structural and regulatory proteins • preparation of cells for S phase - synthesis of replication proteins and histones • normal metabolic activity • length depends on the cell type, availability of nutrients and growth factors, the presence of inhibitors of proliferation • In optimal conditions, 9-11 hrs • the genetic material in the form of decondensed chromatin, 2n DNA molecules (46 in humans) • restriction point is sensitive to external factors, the first cell cycle checkpoint • At the end of the phase, the cell decides how it will proceed i) entry into the G0 phase: resting phase, cell does not divide the absence of growth factors, senescence, differentiated cells ii) entry into S phase: after overcoming the restriction point, the cell continues by next cell division 9-Cell cycle 2022 15 S phase semi-conservative DNA replication • within the replicon based in places ori bidirectional replication fork • replication proteins - initiating proteins, helicases, polymerases, RNases, ligases, telomerase • DNA synthesis requires a template DNA strand and the RNA primer • leading strand is formed continuously lagging strand is formed through Okazaki fragments • replication of the ends of linear chromosomes is ensured by telomerase correctness of replication is ensured by accuracy and proofreading DNA polymerase activity, error base pairing repair • duplication of genetic material, chromosomes composed of sister chromatids 9-Cell cycle 2022 16 G2 phase second growth phase, premitotic phase • protein synthesis and formation of organelles, systematic preparation for M phase • synthesis of proteins of mitotic spindle, centriole duplication • replicated genetic material, 2 x 2n DNA molecules, two sister chromatids in chromosome • 2nd cell cycle checkpoint, checking the integrity and accuracy of DNA replication Centriole • paired organelle of eukaryotic cell, not in plant and fungi cells • capable of independent division • nine microtubule triplets around the central cavity • two connected perpendicularly oriented in M and G1 phase • duplication in S and G2 phase, in the resulting pair there is always one original centriole and one new • together with its surroundings form the centrosome, which organizes the mitotic spindle 9-Cell cycle 2022 17 M phase It includes nukleus division (mitosis, karyokinesis) and the cytoplasm (cytokinesis) • based on changes in morphology and behavior of chromosomes mitosis is divided into 5 phases Interphase Mitotic prophase Mitotic promethaphase Mitotic metaphase Mitotic anaphase Mitotic telophase MITOSIS 9-Cell cycle 2022 18 Early mitotic prophase Late mitotic prophase (prometaphase) Prophase a prometaphase chromosome condensation occurs Outside the core begins to rise the mitotic spindle M-phase 1) Prophase • condensation of chromosomes to form visible under light microscope • sister chromatids connected in centromeric region • centrosome shift to opposite sides of the cell • the formation of the mitotic spindle begins in centrosomes 2) Prometaphase (late prophase) • formation of the kinetochores on chromosomes - A large protein complex in centromeric region - Allows to bind chromosomes to spindle microtubules - Inner layer recognizes and binds to centromere DNA - An intermediate layer connects the outer and inner layer - The outer layer is connected to the (+) ends of microtubules, comprising contains motor proteins, based on the fibrous corona 9-Cell cycle 2022 19 M-phase prometaphase disintegration of the nuclear membrane and nucleolus • stabilization of centrosomes position on opposite sides of the cell • movement of chromosomes into the center of the cell • formation of the mitotic spindle - Tubulin polarity: (-) end in the centrosome, (+) end grows at the opposite end - Dynamic structure, shortening and lengthening (de) polymerization of tubulin - After the disintegration of the nuclear membrane (+) the ends of the spindle contact the kinetochore - Kinetochore microtubules: connected to kinetochores - Polar microtubules: connected to tubules from the opposite pole of the spindle - Astral microtubules: attached to the plasma membrane proteins 9-Cell cycle 2022 20 Chromosomes are grouped in the equatorial plate and form a metaphase plate. M-phase Metaphase Chromosome condensation is completed • chromosomes are pulled / pushed by kinetochore microtubules • sorting of chromosomes in the equatorial plate between the poles of the mitotic spindle (metaphase plate) • sister chromatids of each chromosome are connected to opposite poles of the spindle • establishing the karyotype from the cells which were stopped in metaphase (colchicine) 9-Cell cycle 2022 21 Sister chromatids split and migrate towards opposite ends of the cell M-phase Anaphase • chromosomes separate at centromeres, sister chromatids split • chromatids move towards spindle - kinetochore microtubules shorten - spindle poles become more distant - participation of motor proteins • the same set of genetic information is assembled at each spindle pole, establishing a base for daughter cells Chromosomes move towards spindle apparatus Spindle poles become more distant 9-Cell cycle 2022 22 New nuclear envelope is created around each set of chromosomes to produce two daughter nuclei M-phase Telophase Polar microtubules continue to lengthen, preparing for cytokinesis • chromosomes, which arrived to the poles of the spindle begin to decondense • kinetochores and mitotic spindle deteriorate, new nuclei forms • nuclear membrane restore around each set of sister chromatids9-Cell cycle 2022 23 Meiosis Asexual reproduction • cell divides to produce two new identical daughter cells • e.g. mitosis, binary fission Sexual reproduction • two cells fuse to produce a new cell (zygote), that is different from original cells Meiosis • reduction division (chromosomes) • occurs in germ cells, produces gametes (eggs, sperm) • chromosomes replicate in interphase, there are two meiotic divisions • the original cell is diploid (2n) and produces four daughter haploid (n) cells • chromosome number is reduced by half, fertilization restores the diploid status Diploid – 2n 46 dsDNA Diploid – 2n 92 dsDNA haploid – n 46 dsDNA Homologous chromosome pair Sister chromatids Four gametes (haploid, n, 23 dsDNA) DNA replication Meiosis I Meiosis II 9-Cell cycle 2022 24 Meiosis • occurs in germ cells, produces gametes (eggs, sperm) Spermatogenesis oogenesis Spermatogonium (diploid) Oogonium (diploid) Spermatids (haploid) Sperms (haploid) Polar bodies (haploid) Oocyte (haploid) 9-Cell cycle 2022 25 Meiosis I – reduction division • homologous chromosomes with different alleles for the same genes separate • unusual cell division type, that creates haploid cells with chromosomes originating from sister chromatids Prophase I • homologous chromosomes pair up, bivalent assembly (2 chromosomes), tetrads (4 chromatids) • crossing-over (homologous recombination) takes place between homologous chromosomes connected by a synapse - genetic recombination in offspring is ensured by replacing parts of chromosomes • chromosomes condense, spindle apparatus forms, nuclear membrane disintegrates • consists of five phases Sister chromatids (same genes, same alleles) Homologous chromosomes (same genes, different alleles) Gene X Synapse begins Recombined chromosomes Synapse disintegrates, visible chiasma Synapse finished, crossing over Duplicated chromosomes condense Leptotene Zygotene Pachytene Diplotene Diakinesis 9-Cell cycle 2022 26 Meiosis I – reduction division Metaphase I • chromosome homologous pairs assemble in equatorial plane Anaphase I • homologous chromosomes separate and move towards spindle apparatus • sister chromatids remain connected by centromeres Telophase I • spindle apparatus disintegrates, nuclear membrane restores • cytokinesis splits the cell into two Interphase Prophase I Metaphase I Anaphase I Telophase I Chromosomes replicate Homologous chromosomes pair up, crossing over Tetrads assemble in equatorial plane Homologous chromosomes split up Two haploid cells, chromosomes still doubled Nuclear membrane chromatin Sister chromatids tetrads Centromere with kinetochore Homologous chromosomes separate spindlecentrosomes Kinetochore microtubules Sister chromatids untouchedMetaphase plate chiasma Cleavage furrow 9-Cell cycle 2022 27 Meiosis II – separation division • just one chromosome homologue is present in cell after meiosis I • during meiosis II sister chromatids, that contain matching information separate into newly forming gametes Prophase II nuclear membrane disintegrates, spindle apparatus forms Metaphase II chromosomes align in equatorial plane Anaphase II sister chromatid separation and their movement towards opposite poles of spindle apparatus Telophase II spindle apparatus disintegrates, nuclear membrane restores, cytokinesis Sister chromatids (same genes, same alleles) Gene X Two haploid cells, chromosomes still doubled Sister chromatids Sister chromatids split during meiosis II. Four haploid cells containing a copy of each chromosome arise. Telophase I Prophase II Metaphase II Anaphase II Telophase II Cleavage furrow 9-Cell cycle 2022 28 • sexual reproduction is a source of genetic diversity stemming from: i) unbiased chromosome choice: chromosomes split randomly in meiosis I ii) random fertilization: random fertilization of an egg by a sperm resulting in zygote iii) crossing-over: new allele combinations coming from parent chromosomes Meiosis outcome in four haploid cells, each with a copy of every chromosome • crossing-over generates altered allele combinations within chromosomes • meiosis is the basis for sexual reproduction, depends on sex chromosome presence • offspring produced by sexual reproduction is genetically different from its parents and siblings Mitosis Meiosis DNA replication During interphase, before mitosis During interphase, before meiosis, only once Number of divisions 1 2 Homologous chromosome linked by a synapse Does not occur Occurs together with crossing-over between nonsister chromatids in prophase I Daughter cell number and their genetic content Two diploid (2n) daughter cells, genes identical with parent cell Four haploid (n) daughter cells, genes different from parent cell Role in human body Growth, repair Genetic diversity in sexual reproduction 9-Cell cycle 2022 29 Regulating cell cycle decision whether to split is a cellular reaction to its size and extracellular signals (nutrients, growth factors, stress factors, DNA damage) • yeasts decide mainly according to cell size and nutrient availability • mammals adjust according to growth hormones and mitogens Principles in cell cycle regulation • mitosis wont happen before DNA replication finishes, DNA replication wont happen without splitting the cell • damaged DNA must not replicate and be passed onto daughter cells • erroneously paired chromosomes must not finish mitosis • cell cycle deregulation can lead to carcinogenesis Healthy tissue: organized cell growth cancer: uncontrolled cell growth 9-Cell cycle 2022 30 Cell cycle regulation elements Foundation of this regulation lies in specifically activating proteins that regulate cell cycle i) CDK (cyclin dependent kinase) Posttranslational protein modifications are a part of cell cycle regulations - phosphorylation: major change to protein structure and function, reversible proteinkinases are among the main cell cycle regulators - proteolysis: unneeded proteins are labelled by ubiquitin and decomposed in proteasome, irreversible degradation system ii) Cell cycle checkpoints kinases of the CDK group, cyclines, cyclin inhibitors 9-Cell cycle 2022 31 CDK – cyclin dependent kinases heterodimer proteinkinases with a regulatory (cyclin) and catalytic (kinase) component (cyclin dependent kinase, CDK) They phosphorylate key regulator proteins and are thus involved in cell cycle management 9-Cell cycle 2022 32 Systems for protein degradation: • targeted degradation of proteins regulating cell cycle, results in unidirectional irreversible cell phase shift • Ubiquitin ligase: • SCF • „anaphase promoting complex“ = cyclosome (APC/C) • Ensures labelling targeted protein by polyubiquitin mark, that predetermines proteins for degradation in proteasome is a multi-protein E3 ubiquitin ligase complex catalyzing the ubiquitination of proteins destined for proteasomal degradation. It has important roles in the ubiquitination of proteins involved in the cell cycle and also marks various other cellular proteins for destruction 9-Cell cycle 2022 33 Ubiquitin-proteasome system Ubiquitin activation • Catalyzed by ubiquitin-activating enzyme E1 • ATP dependent reaction • Ubiquitin is attached by C-terminus to E1 protein • Ubiquitin is consequently carried over from E1 onto ubiquitin-conjugating enzyme E2 • Enzyme E2 works in conjunction with E3 enzyme, this complex is termed ubiquitin-ligase 9-Cell cycle 2022 34 Proteasome degradation: major targets • Cell cycle and growth regulators: • Components of cell signal cascades • transcription factors • metabolism enzymes • protein defective variants resulting from mutations or faulty proteosynthesis • major histocompatibility complex I antigens • Proteasome inhibition is lethal to the cell 9-Cell cycle 2022 35 • Mammals posses hundreds of ubiquitin-ligases, that recognize degrons (signals for degradation) on target proteins by recognition section E3 • Ubiquitin-ligases then manage the ubiquitination itself, producing polyubiquitin chains attached in several steps to target protein or previous ubiquitin at lysine residue 9-Cell cycle 2022 36 • Binding of poly-Ub labeled substrate to Ub receptor subunit of the proteasome • Cleavage of Ub through izopeptidase bound to hatch • substrate translocation to the proteasome column • degradation of the substrate into small peptides Degradation in the proteasome 9-Cell cycle 2022 37 9-Cell cycle 2022 38 Cyclin-dependent protein kinases (CDK) • key enzymes which regulate the transition between cell cycle phases, transfer cells from the G0 to G1 • various phases ofCC BC triggered by phosphorylation of specific target proteins • their activity is strictly regulated i) Catalytic activity only in complex with cyclin ii) Tyr phosphorylation sites – activating, inhibitory - phosphorylation by specific kinases, dephosphorylation by phosphatases iii) inhibitors • in yeast one enzyme, in humans five enzymes G1 CDK4, CDK2, CDK6 S CDK2 G2 CDK2, CDK1 M CDK1 • negative regulators of CC passage, two families (INK, CIP) • They bind to CDK and inhibits their activity, responsible for the arrest in G1 • e.g. p21 is actively synthesized during DNA damage, binds to CDK and interrupts the progress of CC TEST 9-Cell cycle 2022 39 Cyclins • proteins that form complexes with CDK, activate Cdk and determine their substrate specificity • regulate the passage of the phases of CC • its concentration fluctuates during the CC phases - specific synthesis / ubiquitination and proteolysis Cyclins of G1-phase (D): Participation in the regulation of cyclins G1 / S Cyclins of G1/S-phase (E): CDK activation in late G1, contributing to overcome the restriction point Cyclins of S-phase (A): CDK activation point after the passage of restriction poinz, on duplication of chromosomes, the S phase cyclin remains elevated until mitosis, because they contribute to the regulation of early mitosis. Cyclins of M-phase (B): Cdk activation stimulating entry into mitosis after passing through the checkpoint G2 / M, degradation in the middle of phase M. 9-Cell cycle 2022 40 The activity of cyclin-cdk complexes - - concentration of cyclins varies during the cell cycle - concentration of CDK is more stable, but their activity is zero without cyclin - cyclins co-decide on the choice of substrate phosphorylation by complex CDK / cyclin 419-Cell cycle 2022 CDK2 E A CDK1 B CDK4,6 http://tbl.med.yale.edu/cell_growth_control/reading.php TEST „anaphase promoting complex“ = cyklozom (APC/C) Cyclin CDK Function D, E CDK4, CDK6, CDK2 They make it possible to overcome the point of restriction in the late G1 A, E CDK2 They allow you to start DNA replication A CDK2, CDK1 They guide the cell to the M phase B CDK1 They support mitotic processes Phase of CC G1 S G 2 M Cyclins • complexes with Cdk, activate Cdk and determine their substrate specificity • their concentration fluctuates during BC phases - specific synthesis / ubiquitination and proteolysis Factors ensuring cell cycle management TEST • in humans 4 families Proteazom CDK Cyklin http://www.bioscirep.org/content/30/4/243 http://tbl.med.yale.edu/cell_growth_control_reading.php https://www.boundless.com/biology/textbooks/boundless-biology-textbook/cell-reproduction-10/control-of-the-cell-cycle-89/regulator-molecules-of-the-cell-cycle 42 9-Cell cycle 2022 439-Cell cycle 2022 TEST 9-Cell cycle 2022 44 9-Cell cycle 2022 45 9-Cell cycle 2022 46 Three functional states of Cdk A) inactive - no cyclin - Cdk active site is blocked by the portion of the protein, called T-loop (B) partially active - after binding of cyclin T-loop leaves the active site of Cdk and partially activates it (C) fully active - threonine phosphorylation of the T-loop Cdk kinase by CAK (cdk-activating kinase): conformational changes, increased affinity for the respective substrates 9-Cell cycle 2022 47 The regulation of CDK activity: a) phosphorylation binding of cyclins primarily determines the activity of Cdk assistance mechanisms contribute to the fine regulation of CDK activity: - phosphorylation of pair of amino acids near the active site of the kinase Wee1 inhibits the activity of cyclin / CDK complexes - dephosphorylation of amino acids by phosphatase Cdc25 enhancing activity of cyclin / CDK complexes - other activating phosphate adds CAK to T-loop 9-Cell cycle 2022 48 • bind to cyclin / CDK complexes and inhibit in • due to changes in conformation of the active site in Cdk The regulation of CDK activity: b) inhibitory proteins CKIs a) family Cip/Kip b) family INK4 9-Cell cycle 2022 49 CDK inhibitors: Cip/Kip family three members in mammals: p21 Cip1 , p27 Kip1 , p57 Kip2 distinct roles in cell cycle regulation p21 is induced by DNA damage using p53 - ensures arrest of cell cycle in G1 and G2 phases p27 applies when leaving the cell cycle when they accumulate; upon re-entry into the cell cycle from dormancy is rapidly degraded p57 is applied during embryogenesis, is expressed tissuespecifically during development 9-Cell cycle 2022 50 CDK inhibitors: INK4 family (“inhibitors of CDK4“) p16 INK4A p15 INK4B p18 INK4C p19 INK4D Common signs: inhibiting the formation of a complex of cyclin D and CDK4 / CDK6 and inactivation of already formed complexes cell cycle arrest in the G1 phase (prior to the restriction point) • expression of p16 stimulated by growth inhibitors (e.g. TGFβ) and E2F (negative feedback) 9-Cell cycle 2022 51 Pharmacological inhibitors of CDKs • CDKs contol cell cycle, control gene expression, differentiation, apoptosis, processes in the nervous system • deregulation of CDKs in diseases, pharmacological inhibitors blocking the ATP binding site of CDK • investigation of the selective action of these inhibitors and their possible use for medical purposes • - tumors, neurodegenerative diseases (Alzheimer's disease, ALS, stroke) • - cardiovascular diseases (atherosclerosis, heart hypertrophy) • - viral infections (HCMV, HIV, HSV, HPV) • - parasitic protozoa (malaria, sleeping sickness) Palbociclib (PD-0332991) (inhibitor of CDK4 and CDK6) gave encouraging results in a phase II clinical trial on patients with estrogen-positive, HER2negative advanced breast cancer 9-Cell cycle 2022 52 CC checkpoints-molecular brakes They control : ✓ initiate S phase ✓ initiation of mitosis ✓ distribution daughter chromosomes in anaphase ✓ beginning of telophase and cytokinesis ✓ DNA integrity ensure that major events of cell cycle tate place in a fixed order sensors monitor key going cycle and provide a feedback control system if the error is logged, the control system will extend the stage that the error could be corrected provide extremely accurate cell division (receiving the correct number of chromosomes correctly replicated by daughter cells) 9-Cell cycle 2022 53 They perceive external and internal stimuli and ensure the proper operation of CC • checking the completion of the previous phase, and fulfillment of conditions for the next phase G1 checkpoint - restriction point • external factors - the presence of nutrients, mitogens, antiproliferative factors • after crossing restriction ponit, CC can be stopped only by internal factors • DNA integrity • size of cells (yeast) G2 checkpoint • completion of DNA replication • DNA integrity • size of cells (yeast) M checkpoint • metaphase-anaphase transition • proper connection of mitotic chromosomes to the spindle CC checkpoints-molecular brakes 9-Cell cycle 2022 54 External stimuli - chemical • animal cells require addition of essential nutrients, extracellular signals for growth, division and survival • signaling molecules produced by other cells of the same organism: i) mitogens - stimulate cell division, overcome natural braking mechanisms G1 / S (e.g. Rb) ii) growth factors - stimulate increase in mass and cell growth, induce the synthesis of macromolecules - e.g. PDGF: formation in platelets, promotion of wound healing iii) factors for survival - suppress programmed cell death External stimuli – physical for growth and proliferation cells require space and surface for adherence 9-Cell cycle 2022 55 Internal stimuli • DNA integrity, connecting chromosomes to the spindle DNA damage due to physical and chemical mutagens • repairable damage - stop of CC to the time of repairing DNA • extensive damage - programmed cell death • CC arrest in G1 (inactivation of CDK2) or G2 (Cdk1 inactivation) • the key role of tumor suppressor genes ATM, ATR - recognition of DNA damage (UV photoproducts, dsDNA breaks), activation of CHK1 CHK2 CHK1, CHK2 – activation of p53, inhibition of Cdc25 p53 - increased levels of CKIs 9-Cell cycle 2022 56 Cell cycle checkpoints p53 deficiency • through the cell cycle with damaged DNA - the accumulation of mutations – risk of tumors (mutations in the p53 gene in the majority of cancer) • Li-Fraumeniho syndrome - hereditary syndrome caused by p53 inactivating mutations - increased risk of cancer (sarcomas, brain tumors, breast cancer, adrenal gland, leukemia) - 50 % risk of cancer up to 40 years of age, 90% to 60 years of age p53 • important cell cycle regulator, acts as a tumor suppressor • Under normal conditions, a low level, continuous degradation - Mdm2 provides transport p53 from the core to the proteasome - Feedback: p53 triggers the expression of Mdm2 • stabilized in stressful situations, especially when DNA damage • acts as a transcription factor, induces - cell cycle arrest: expression of p21/CIP - stop DNA replication: inactivation of DNA polymerase δ - mitosis arrest: inactivates cdc25 - apoptosis: expression of Bax, Puma CHK1, CHK2 demage DNA actiovation activation degradation inhibition CKI Cyklin - CDK No phosphorylation pRb No inhibition apoptosis inhibition TEST 9-Cell cycle 2022 57 Cell cycle checkpoints Spindle checkpoint • for entry into anaphase cells must receive confirmation of the connection of all chromosomes to spindle microtubules • damage to this control mechanism leads to damage and nondisjunction of chromosomes i) checkpoint ON • unattached kinetochore activate Mad and Bub proteins • complex APC-Cdc20 in inactive state ii) checkpoint OFF • Sister chromatid chromosomes binding on microtubules from opposite ends of spindle causes mechanical stress in the chromosomes • complex of proteins Mad and Bub not formed • APC-Cdc20 complex is active and provides securine degradation 9-Cell cycle 2022 58 Mitogenes pro-growth signaling molecules, that activate genes of immediate response by intracellular signaling cascades products of immediate response genes activate genes of delayed reaction (eg. cyclin D) cyclin D/CDK phosphorylates Rb positive feedback: E2F enhances the transcription of its own gene; G1/S-Cdk and S-Cdk increased phosphorylation Rb 9-Cell cycle 2022 59 E2F proteins E2F are transcription factors that activate gene expression encoding proteins necessary for DNA replication, cyclins of late G1 and S phase and CDK of S phase The absence of mitogenes: gene expression controlled by E2F is unabled by interaction between E2F and Rb protein The presence of mitogens: G1-CDK phosphorylates Rb, Rb-E2F bound is released and E2F subsequently ensure expression of target genes 9-Cell cycle 2022 60 Crossing point restrictions: participation of Rb Rb protein is a key substrate of cyclin-CDK complex of G1 phase phosphorylation Rb in several places prevents its association with E2F phosphorylation Rb is further increased during a cell cycle by phosphatase PP1 Rb passes to the hypophosphorylated state of G2 phase 9-Cell cycle 2022 61 Factors influencing the passage of the cell cycle presence of mitogenes – expression of cyclin D • presence of antimitogene, differentiation factors – expression of CKI • cell aging – expression of CKI • damage of DNA – arrest of cell cycle through p53 • viral infections (eg. HPV, human papillomavirus) – inhibition of p53 and pRb, cell proliferation DNA damage 9-Cell cycle 2022 62 Cell cycle control – early phase of G1 • cyclin D - CDK4/6 • Three forms of cyclin D (D1, D2, D3) with tissue-specific expression form complex with CDK4 or CDK6 • cyclin D acts as a sensor of mitogenic signals and prepares the cell for the S-phase • activation of transcription factors, which provide expression of replication proteins and cyclin E • dephosphorylation of components pre-initiation complexes, building of new complexes in places ori • response to mitogenic signals: eg. EGF - EGFR - GEF factor - Ras protein - MAP kinase pathway transcription factors – expression of cyclin D • inhibition of antimitogenes signals eg. TGFβ - TGFβR - transcription factor Smad3, Smad4 – expression of CDK inhibitor mitogen signalisation antimitogen signalisation 9-Cell cycle 2022 63 Cell cycle control – transition of G1/S cyclin D - CDK4/6, cyclin E - CDK2 • further prepare the cell to the S phase • transcription of cyclin E controlled by a set of transcription factors E2F (activators 1-3, inhibitors 4-5) • Rb protein (pRb) active in unphosphorylated form, inhibited by phosphorylation • in G0 and G1 pRB inhibits by its binding activating E2F and activates E2F inhibition, suppresses the expression of cyclin E • after phosphorylation by complex cyclin D - CDK4/6 pRb is released and E2F triggers expression of cyclin E, cyclin A and replication proteins • Positive feedback – cyclin E formes complex with CDK2 and complets pRb phosphorylation, at this point it is through the cell cycle independent of the presence of a mitogen • after initiation of S phase, cyclin E is degraded and CDK2 forms a complex with cyclin A 9-Cell cycle 2022 64 Cell cycle control – S phase cyclin A - CDK2 • phosphorylation of components of the pre-initiation complex - initiation of DNA replication (activation complex MCM) - block of repeat replication within a cell cycle (cdc6 releasing) • The result of S phase is the emergence of two sister chromatids of each chromosome associated by cohesin molecules 9-Cell cycle 2022 65 Cell cycle control – transition of G2/M MPF (mitosis-promoting factor) = cyclin B - CDK1 • cyclin B concentration increases continuously throughout interphase • CDK1 - inhibitory kinase phosphorylation by Wee1 removed by phosphatase Cdc25 - activating phosphorylation by kinase CAK • increase in MPF activity is jumping, at maximum level of cyclin B and after removal of inhibitory phosphorylation • Positive feedback - activated MPF activates Cdc25 and inhibits Wee1 MPF phosphorylates target proteins involved in early stage of mitosis • chromatin condensation - activation of condensins • disintegration of the nuclear membrane – depolymerization of laminates, phosphorylation of nuclear pore and proteins of inner membrane • formation of the mitotic spindle - activation of centrosomes and proteins associating with microtubules Control of cell size in yeast cells • low activity of Cdc25 and Wee1 high activity - delayed mitosis, prolonged G2, cell enlargement • high activity of Cdc25 and low activity of Wee1 - Fast Track mitosis, shorter G2, reduce cell 9-Cell cycle 2022 66 Cell cycle control- M phase APC, anaphase-promoting complex • ubiquitin ligase connecting ubiquitin to proteins that control mitosis, and thus predetermines degradation • activity during the cell cycle fluctuates, activated in late mitosis by treatment MPF • only works when connected Cdc20 protein, the protein is blocked by Mad and Bub, who released him up after connecting the kinetochore of chromosomes to spindle microtubules • substrate are e.g.: i) securin • its decomposition relaxes separase which degrades proteins linking sister chromatids ii) cyclin B • a sudden drop in his level inactivates MPF • absence of MPF allows constitutively active phosphatases to remove phosphate groups from proteins that were phosphorylated MPF, mitotic endings, entry into a new G1 9-Cell cycle 2022 67 Disorders of cell cycle control Tumour diseases • tumor cells do not need to divide the growth factors - themselves can generate growth factors - May signal in the absence of growth factor - May have impaired cell cycle checkpoints • increased proliferation of cells to support its transformation into tumor cells • tumor is basically failure to control cell division, uncontrolled cell growth • protooncogenes - normally activate cell proliferation - an abnormal activation or losing of inhibition - support tumorigenesis - eg. growth factors, cyclines, CDK, E2F, Mdm2, Ras, Myc • tumor suppressors - normally suppress cell division - inactivation or increased inhibition promotes tumor formation - eg. pRb, p53, p21, p14, p16, TGF-β, ATM 9-Cell cycle 2022 68 Disorders of cell cycle control Numerical chromosomal abnormalities (aneuploidy) • normal karyotype 46, XX(Y) monosomy - eg. 45, X trisomy - eg. 47, XY +21 • Errors in the mitotic spindle checkpoint, the result of delays and non-disjunction of chromosomes i) Errors in mitosis • chromosome delays in anaphase and non-inclusion in the core subsidiary • formation of mosaicism, in one organism occurring cell lines with different karyotype ii) Errors in meiosis • non-disjunction - creation of nulisomic/trisomic gametes monosomy/trisomy after fertilization - in MI: error in the distribution of homologous chromosomes - in MII: error in the chromatids division • chromosome delays in anaphase : the formation of nulisomic gamete 9-Cell cycle 2022 69 9-Cell cycle 2022 70