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Abnormal Cell Proliferation,Differentiation Apoptosis and Related Disease Department of Pathophysiology Shanghai Jiao-Tong University School of Medicine The Cancer Problem Evan, GI and Vousden, K. (2001) Nature 411:342 Proliferation Differentiation Cell Apoptosis Abnormal Cell Proliferation and Diseases 【Cell Proliferation】 Cell proliferation is a process of cell division and regeneration, which results in an increase in the cell number with exact passages of genetic information to their daughter cells. Phase of cell cycle • M-Phase – Mitosis – Cytokinesis • Interphase – G1 = Gap between M and S – S = Synthesis (DNA and centrosomes replicated) – G2 = Gap between S and M Mitosis Interphase Anaphase Prophase Prometaphase Telophase Cytokinesis Metaphase 2001 Nobel Prize Leland H. Hartwell 1970s “Checkpoint” Yeast genetics ~100 CDC genes Start gene Paul M. Nurse 1970s CDKs yeast Tim Hunt 1980s Cyclins Sea Urchins Cell Cycle Regulation • Cyclins • Cyclin Dependent Kinases (CDKs) • Cyclin Dependent Kinases Inhibitors (CDKIS) ●Cyclin 【Classification 】 Cyclin B1、Cyclin A、Cyclin E、Cyclin D1,D2,D3. F,G,H,T. Cyclin Protein kinase process regulated Cyclin D1-3 Cdk 4,6 G1-phase progression Cyclin E Cdk2 G1 to S-phase Cyclin A Cdk2 S-phase progression Cyclin A Cdk1 S through G2 Cyclin B Cdk1 M-phase Cyclic Degradation: Ubiquitin-mediated breakdown by Proteasome Experimental Demonstration that Cyclin D is Required for Passage Through the Restriction Point in the Mammalian Cell ●CDKs CDKs are group of serine and threonine kinases. 【CDK1 activates cell cycle】 Thr14、Tyr15 dephorsphorylation Cyclin B binds CDK1 CDK1activation phosphorylation of CDK1 by CAK Cell cycle CDK activation kinase (CAK) 【CDK activation and cell cycle regulation】 ▲ CDK2 binds cyclin E, CDK4、CDK6 binds cyclin D1、D2、D3 pRb phosphorylation ▲ E2F release↑ initiate G1 phase DNA synthesis initiate S phase CDK2 binds cyclin E and A recognize chromosome ARS Accelerate S phase ▲ CDK1 binds cyclin B1 initiate M phase ▲ CDK1 binds cyclin A initiate G2 phase ●CDI Ink4:P16INK4a、P15INK4b、P18INK4c和P19INK4d ▲ Cip/Kip:p21Cipl、P27Kip1和P57Kip1 ▲ 【mechanism of inhibition】 CDIs direct bind the phosphorylation site of CDKcyclin complex. ▲ P16INK4a binds CDK4, inhibits its interaction with cyclins. ▲ CDK2 CDK4、CDK6 ■Checkpoint of cell cycle) DNA damage Checkpoint G1/S DNA replication S/G2 spindle assembly G2/M 【Check mechanism】 ●P53 :In response to DNA damage in G1, the protein level of p53 rises dramatically due to the activation of a checkpoint protein kinase (chk2) that phosphorylate p53 and make it less susceptible to degradation by the ubiquitination/proteasome pathway. This enables p53 to activate transcription of the cdk inhibitor (CKI) p21 which binds to all cdks and inhibits their action and thus “arrests” the cells until the DNA damage can be repaired. DNA damage, other insults Growth Signal p53 p21 G1 phase cyclin/CDK S phase ►Abnormal cell cycle & diseases Deregulation of cell cycle causes cell proliferation excessive or insufficency. 【Deregulation of cell cycle】 ■loss ■The of control in driving cell cycle progression(Cyclin,CDK,CDI) impairment of checkpoint system Abnormal proliferation differentiation related disease abnormal proliferation abnormal differentiation abnormal proliferation and differentiation aplastic anemia leucoderma obesity malignant tumor heritage hemoglobinopathy prostatic hypertrophy muscular dystrophy scleratheroma Familial erythrocytosis psoriasis dysembryom congenital deformity X-linked hyper IgM syndrome Malignant tumor Cancer is a disease where regulation of the cell cycle goes awry and normal cell growth and behavior is lost . 1. loss of control in driving cell cycle progression ■ Overexpression of cyclins 【Cyclin D1 overexpression】(oncogene) ▲gene amplification(major reason) Observed in breast, gastric, esophageal cancer. ●Chromosome rearrangement Cyclin D1 gene under the control of parathyrin promoter Synthesis of Cyclin D1 increase ●Chromosome Parathyroid carcinoma translocation t(11:14) (q13:q32) Cyclin D1 gene under the control of Ig heavy chain gene enhancer Cyclin D1 overexpression ▲gene mutation Cyclin D1 T286mutation Cyclin D1 ubiquitination block Cyclin D1↑ 【mechanism of Cyclin D in tumorgenesis】 Cyclin D overexpression + cytokines CDKs water fall effect↑ cell over proliferation susceptible to cancerization ■ abnormal expression of CDK Mainly observed in CDK4、CDK6 overexpression. CDK4↑+ cyclin D binding↑ pRb CDK4/cyclin D↑ CDKs expression↑ pRbphosphorylation↑ cell over proliferation E2F ↑ G1/S transition accelerated ■ CDI insufficency and mutation ▲ InK4 inactivation 【causes of p16 InK4 inactivation】 mutation or deletion, chromosome translocation, hypermethylation. 【Mechanism】 p16 InK4 gene expression↓ CDK4/cyclin D binding↓ cell susceptible to proliferation susceptible to tumorgenesis ▲ Deficient expression of Kip/Cip P21cipl function PCNA cyclins/CDKs activity↓ cell cycle DNA replication block 【Mechanism】 p53 mutation P21cip1 transcription↓ DNA damage cell↑ 2. Impairment of checkpoint system main reason:G1/S、G2/M checkpoint dysregulation consequence:function of detect DNA damage Table 8.3 The Biology of Cancer (© Garland Science 2007) Table 8.4 The Biology of Cancer (© Garland Science 2007) ■G1/S transition dysregualtion ▲P53 enter S phase DNA damage G1/S check successful G1 arrest DNA repair failure apoptosis ▲P53 mutation or loss bcl-2 bax P53 susceptible to cancergenesis mutagen checkpoint function↓ genetic instability replication fidelity↓ Table human tumor p53 gene mutation hotspot and frequency tumor frequency(%) hotspot tumor frequency(%) hotspot Lung cancer 56 157,248,273 prostatic caner 30 unknown Colon carcinoma 50 175,245,248,273 hepatocellular carcinoma 45 249 Esophageal carcinoma 45 unknown colloid carcinoma 25 175,248 Ovarian cancer 44 273 breast cancer 22 175,248,273 Pancreatic cancer 44 273 endometrial cancer 22 248 Skin cancer 44 248、278 thyroid cancer 13 248,273 Gastric cancer 41 unknown leukemia 12 175,248 Head and neck squamous cell carcinoma 37 48 uterine cervix cancer 7 273 Bladder cancer 34 280 soft tissue sarcoma 31 unknown DNA virus: SV40、HPV、adenovirus P53 inactivation ■ impairment of G2/M checkpoint G2/M transition DNA double strand break activation of DNA damage checkpoint block cell enter M phase induce repair gene transcription DNA repair ▲ loss of G2/M checkpoint chromosome rearrangement, loss Primary Throbocythemia symptoms :is characterized by a platelet count greater than 600,000/µL, megakaryocytic hyperplasia, splenomegaly, and a clinical course complicated by thrombotic and/or hemorrhagic episodes. ■ clinical ■ The cause of ET is not fully understood. About half of patients with ET have a mutation of the JAK2 (Janus kinase 2) gene in their blood cells. Whether or not a patient has the mutation does not appear to affect the nature or course of the disease. Research is under way to determine the precise role of JAK2 mutations and to identify other mutations in ET patients. Apoptosis and Diseases 【Concept of apoptosis】 Apoptosis is a genetically controlled process regulated by complex molecular signaling systems. It is also known as cellular self destruction or cell-suicide or programmed cell death (PCD). 【physiology implication】 ■ is involved in tissue development ■ maintain ■is the homeostasis involved in defense response Physiology or Medicine Nobel Prize 2002 1090 –131= 959(cells) Robert Horvitz John Sulston Sydney Brenner “for their discoveries concerning genetic regulation of organ development and programmed cell death” Features of apoptosis cells 1. The morphologic characteristics of apoptosis ■ loss of contact with neighbering cells ■Blebbing of cell membrane ■Condensation ■Apoptotic APL of chromatin bodies Apoptosis apoptotic cell (SEM) (TEM) Morphological features of apoptosis Membrane blebbing 2. The biochemical characteristics of apoptosis 1)DNA ladding pattern activation of endonuclease cleave DNA at specific sites between nucleosomes oligonucleosomal fragments (160~200bp intervals) 2)Phosphatidylserine externalization 3)activation of caspase and its function Caspase (cysteine-containing aspartate-specific protease) Stimuli of apoptosis Table:effects of various factors on apoptosis Inducers Inhibitors Physical and Irradiation, high chemical factors temperature, stress, chemotherapeuti c drugs cytokines IL-2、NGF Hormone and cytokines Glucocorticoid, TNF hormone ACTH, testosterone, estrogen Immunity granzyme others Zn2+、agrypnal、cystein protease inhibitor, EBV, cowpox, virus, neutral amino acid pathogen HCV, HIV Mechanism of apoptosis Caspase-dependent apoptosis (1) Concept, Classification, Construction of Caspases ■ Caspase belong to cystein proteases ■ Caspase categories ▲ Caspase Death receptor pathway upstream(Caspase-8 , -9, -10) Mitochondrial pathway (Ced-3 subfamily)downstream(Caspase ■ -2, -3,-6,-7)(common pathway) Caspase activation Pro-caspase: NH2 terminal domain、 20KD subunit、 10KD subunit cleavage Interaction of large and small subunit (dimer) caspase activation(tetramer) 1. Extrinsic or cytoplasmic pathway ■Death receptor TNFR, FasR, DR3, DR4, DR5, The DR family is part of the tumor necrosis factor receptor superfamily. Triggering members of the DR family by death ligands results in the transduction of either apoptotic or survival signals. Growth factors, Fas antibody ■pathway FSAL FasR DD FADD DED DED Pro-caspase-8 Caspase-8 apoptosis Pro-caspase-3 Caspase-3 FADD(Fas-associated death domain); DD(death domain); DED (death effector domain)。 2. Intrinsic or mitochondrial pathway Bid Ca2+ NO hypoxia ROS stress Caspase-8 MPT open Apaf-1 Cyt.c + ATP MMP↑、swelling、△m↓ AIF Apoptosome Cyt.c caspase-independent apoptosis Apaf-1activation Cyt.c Apaf-1activation CARD + Pro-caspase-9 Caspase-9 apoptosis Pro-Caspase-3 Caspase-3 3. Activation of caspase-3 (Effector caspase) Pro-caspase-3 caspase-9 Caspase-3 Pro-caspase-9 Pro-caspase-6 Caspase-6 caspase-7 Pro-caspase-7 caspase-2 CAD Cleavage of structural protein Pro-caspase-2 ICAD DNA cleavage apoptosis Non-Caspase dependent apoptosis ■calcium overload Activation of Ca/Mg2+dependent endonuclease, DNA cleavage;activation of transglutaminase, apoptotic bodies. ■AIF Mechanism of calcium overload induced apoptosis TNF-α、CD3-Ab、TCDD Plasma [Ca2+] ↑ ■activation of Ca2+/Mg2+dependent endonuclease,DNA cleavage; ■activation of transglutaminase, wide cross-linking of cytoskeleton. ■transcription of apoptosis related gene; ■ mitochondrial dysfunction apoptosis Mechanism of apoptosis 1.Oxidation damage Oxygen free radicals disrupt the normal redox equilibrium, result in the injuring of protein,lipid and DNA. 【Mechanism】 Activation of p53, consuming of ATP, membrane lipid peroxidation, activation of endonuclease, initiating apoptosis. 2.Calcium overload 3.Mitochondria damage Regulators of apoptosis Table 1 apoptosis regulators function gene inhibitors Bcl-2 Bcl-XL、A1/Bfl-1、Bcl-w、Bcl-G and Mcl-1 activators P53、Bax、Bad、Bak、Bid、Bim、Bik、Bok、 Bcl-B、Bcl-Xs、Krk、Mtd、Nip3、Nix、Noxa Dual regulators c-myc、Bcl-x 1.Bcl-2 Family & regulating functions ■ Bcl-2(Bcl-XL)inhibit MPT open,Cyt.c 、AIF release↓ ■ Bcl-2 binds and inactivates Apaf-1,blocks caspase-9 activation ■ Bcl-2 binds cyt C Bcl-2 MPT open Apaf-1 Cyt.c + ATP MMP↑、swelling、△m↓ AIF Apoptosome Cyt.c caspase-independent apoptosis Apaf-1activation Cyt.c Apaf-1activation CARD + Pro-caspase-9 Caspase-9 apoptosis Pro-Caspase-3 Caspase-3 2.p53(tumor suppressor gene) As a transcription factor, p53 regulates downstream genes important in cell cycle arrest, DNA repair, and apoptosis. After DNA damage, p53 holds the cell at a checkpoint until the damage is repaired. If the damage is irreversible, apoptosis is triggered. 【Mechanism】 ■ Downregulates the expression of Bcl-2. ■ P53 induces Bax, NOXA, PUMA expression. ■ P53 induces Fas expression. ■ P53 relocalizes Fas to cell membrane. Table . Human diseases associated with disordered apoptosis Decreased Apoptosis Increased Apoptosis Epithelial Tissue Carcinogenesis Macrophage s Bacillary dysentery Blood Vessels Intimal hyperplasia Myocardium Peri-infarct border zones Lymphocytes Autoimmune disorders Lymphocytes lymphocytes depletion in HIV injections and sepsis Haemopoeitic systems Leukemia, lymphoma CNS Neurodegenerative diseases like Alzheimer’s and Parkinson’s disease Apoptosis inhibition 1. Tumor ■ bcl-2 overexpression In follicular lymphoma, a chromosomal translocation commonly occurs between the fourteenth and the eighteenth chromosomes-t(14;18)-which places the Bcl-2 gene next to the immunoglobulin heavy chain locus. This fusion gene is deregulated, leading to the transcription of excessively high levels of bcl-2. This decreases the propensity of these cells for undergoing apoptosis. (also observed in breast cancer, lung cancer, malanoma, prostate cancer) ■ p53 deletion or mutation Abnormal of P53 in stability, localization or activation p53 expression or function defect apoptosis rate↓ ( abnormal P53 observed in at least 50% malignant cancer) tumorgenesis ■virus oncogene HBV encoded HBX is inhibitor for Caspase-3, and is associated with liver cancer. ■fusion protein Chronic myeloid leukemia(CML) t (9;22)( Ph chromosome) Bcr-abl fusion gene NF-B activation、inhibit apoptosis↓ p210 Bcr-abl fusion protein CML cells proliferation leukemia 2.Autoimmune diseases A common feature of autoimmune diseases is the breakdown of tolerance of self antigens, a consequence of which is the production of autoantibodies reactive with multiple self proteins. Defect in apoptosis can cause autoimmunity by allowing the survival of autoreactive T and B cells. Apoptosis excess 1. Ischemia reperfusion injury ■characters ▲early phase apoptosis,late phase necrosis. ▲in the centre of infarct necrosis,in the peri-infarct zone apoptosis; ▲mild ischemia apoptosis,severe ischemia necrosis; ■mechanism ▲oxidation ▲FasR ▲p53 activation Deregulated cell differentiation and Disease ►Concept Cell differentiation is a process in which a generic cell develops into different kinds cells with the specialized morphology, metabolism and physiological functions in response to specific triggers from the body or the cell itself. Cell differentiation is tightly regulated by a series of regulatory proteins. Ey gene in the morphogenesis of eyes Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors Cell 2007,131,861-872 (Oct3/4, Sox2, Klf4, c-Myc ) Determination and differentiation The determination of different cell types (cell fates) involves progressive restrictions in their developmental potentials. When a cell “chooses” a particular fate, it is said to be determined, although it still "looks" just like its undetermined neighbors. Determination implies a stable change - the fate of determined cells does not change. Differentiation follows determination. In some cases, determination results from the asymmetric segregation of cellular determinants. However, in most cases, determination is the result of inductive signaling between cells. The regulation of cell differentiation 1. Regulation on the genomic level) ■ House-keeping genes House-keeping genes generally encode proteins that participate in basic or universal cellular functions essential for maintaining cell survival. ■tissue specific gene 2. Regulation on transcription and post- transcription levels) ■transcription level ■post-transcription level 3. tranlational and post-translational level 4. Extracellular factors that control differentiation Hematopoiesis “Acute promyelocytic leukemia (AML M3) is now the most frequently curable acute leukaemia in adults if promptly diagnosed and adequately treated.” Parmar S, Tallman MS. , 2003 acute promyelocytic leukemia 1988: Breakthrough in treatment from China 1990: Multiple groups identify RARa as t(15; 17) breakpoint Drug Gene How does ATRA induce remission? How does PML-RARa induce APL? Pulmonary fibrosis Pulmonary vascular lesions myofibroblast hypoxia collagenoblast gas diffusion disorder brinase↑↑↑ collagen deposition alveolar membrane thickening Morbid obesity Excess pre-adipocyte differentiate into adipocyte.