Diseases assotiated with abnomal signal transduction and cellular proliferation and apoptosis

Cell proliferation Cell entered the cell cycle sequentially under the accurate regulation. Significance of cell proliferation 1. Cell renewing 2. Providing resource for cell differentiation 3. Supplement of dead cells Basal concept

Cell apoptosis Apoptosis is a form of programmed cell death in multicellular organisms triggered by external and internal signals under some physio- or pathological conditions. Significance of apoptosis 1. Maintaining homeostasis of the body 2. Facilitating development of organs 3. Removing the damaged cell

One Cell proliferation and apoptosis are regulated by genes. Two Regulation is implemented by gene products of oncogenes and anti-oncogenes. Article 1 Regulation of cell proliferation and apoptosis

1 cell proliferation and its regulation 1. cell cycle definition: a period from the end of one division to the beginning of next division of a proliferative cell. G 0 G 1 SG 2 M 2. Check point of cell cycle G 0 / G 1 G 1 / S G 2 / M 3. Major cell cycle regulatory proteins Cyclin, subtype: A-I, K T Cyclin-dependent kinase, CDK 1-10 CDK inhibitor, CDKI One Gene regulation of cell proliferation and apoptosis INK4 family: P15/16/18/19 CIP/KIP family: P21/27/57

Expression of human cyclins through the cell cycle

Role of CDK in cell cycle cyclin Activity form

2 Proteins involved in cell apoptosis 1. Caspase family 2. Bcl-2 family anti-apoptotic Bcl-2 pro-apoptotic Bax Bak/Bid Bad, et al. 3. Tumor necrosis factor receptor TNFR Fas CD95 TNF-R1 DR4 DR5

Two Regulation of onco-gene and anti-oncogene by signal transduction Normal cells Reproduce themselves exactly, stop reproducing at the right time, stick together in the right place, self destruct if they are damaged or become specialized or mature. Cancer Is a class of diseases in which a group of cells display uncontrolled growth, invasion, and sometimes metastasis resulted from the disorder of cell proliferation, differentiation, aging and death. Mechanisms mutations of onco- and anti-oncogenes caused by mutagens and unstability of genes Onco-gene and anti-oncogene Main genes of regulating cell proliferation and differentiation

DNA mutation causes abnormal cell proliferation and apoptosis Normal cell DNA damage Somatic cell gene mutation Tumor suppressor inactivation Quantitative and qualitative changes of gene products Unbalance in cell proliferation and apoptosis Autoimmune disease (insufficient apoptosis) tumor Degenerative disease in nervous system (over apoptosis) Genetic faults DNA repair gene inactivation Change of appoptosis regulatory gene expression Oncogene activation DNA damage factor Successful DNA repairing DNA repairing failure

Article 2 Oncogene, anti-oncogene and signal transduction mediated by onco- and anti- oncogenes Oncogene Signal transduction associated with cell growth Anti-oncogene (tumor suppressor gene )

One oncogene, onc Basal concept These genes code for proteins that are capable of stimulating cell growth and division. In normal tissues and organisms, such growth-stimulating proteins are regulated, so that growth is appropriately limited. However, changes/mutation in these genes may result in loss of growth regulation, leading to uncontrolled cell proliferation and tumor development. These changed genes are known as oncogenes, because they induce the oncogenic state cancer. Oncogenes are dominant, because a change/mutation of only one allele of that gene can lead to tumor formation.

1 Oncogene 1. Virus oncogene, v-onc These genes are in viruses, may lead to uncontrolled cell proliferation and tumor development. Virus oncogenes are homolog with that corresponding cellular oncogenes. No intron Mutation pattern

Retrovirus Chronic transforming retrovirus wild type) RNA virus: containing gene coding for transcriptase Basic structure and function of retrovirus Genome: Two copies of double strand RNA, 3 9Kb Three structural genes (5-gag--pol--env--3) : gag--- core protein pol---transcriptase env---coat protein Long terminal repeats, LTR promoter enhancer adding signal sequence of polyA

General structure of retrovirus LTR

pol gag env

provirus Infectious course of retrovirus

Jumping replication

Provirus A provirus is a DNA mediate with LTR at both ends that has integrated itself into the DNA of a host cell. Provirusexpressionpackaged into new viral articlebudding outside cell surface Resource of v-onc Definite DNA sequence captured from host cell example: src ALV(no v-src)--------------ASV(containing v-src) ( avian sarcoma virus, ASV )

Obtaining of v-onc Provirus DNA Cell genome DNA Virus DNA DNA Integrating Transcripting, splicing Packaged into virus article

Acute transforming retrovirus Structure features Having different types of defects in structural genes, replication defect, non-infective virus Inserting definite sequence-oncogene Only containing one sort of oncogene Infection features Short latent period Having ability to transforming the corresponding target cells in vitro into malignant cells Exceptions Avian sarcoma virus, ASV Oncogene locates in downstream of structural gene and does not breakdown structural gene of virus. Avian erythroblastosis virus, AEV Containing two sorts of oncogenes: erb-A and erb-B

2. cellular oncogene, C-onc Real copy of v-onc in eukaryotic cell. An cellular oncogene is an activated form of pro-oncogene. A proto- oncogene is a normal gene that can become an oncogene due to mutations or increased expression. Proto- oncogenes code for proteins that help to regulate cell growth and differentiationhouse-keeping genes. Upon activation, a proto-oncogene (or its product) becomes a tumor-inducing agent, an oncogene. Proto-onc In normal cells, c-onc exists in non-activated form.

Features of c-onc (1) Structral features with the general structure of eukaryotic genes ; Different creatures: introns with greater difference exons conservative sequence (2) Widespread in living nature (3) Highly conservative in gene sequence (4) Executing function by products of protein ; (5) Upon activation ( mutations or increased expression ), becomes a tumor-inducing agent, an oncogene.

Differences between v-onc and c-onc V-onc often loses some sequences at both ends No introns in v-onc V-onc is the mutated form of c-onc

Classification of c-onc There are several systems for classifying oncogenes, but there is not yet a widely accepted standard. They are sometimes grouped both spatially (moving from outside the cell inwards) and chronologically (parallelling the "normal" process of signal transduction). Pro-onc mains codes for key molecules involved in signal transduction and gene transcription.

There are several categories that are commonly used: Category Example Growth factors or mitogens c-sis Receptor tyrosine kinases epidermal growth factor receptor(EGFR), platelet-derived growth factor receptor(PDGFR), and vascular endothelial growth factor receptor VEGFR Cytoplasmic tyrosine kinases src-family, syk-ZAP-70 family, and BTK family of tyrosine kinases, the Abl gene. Cytoplasmic serine/threonine raf kinases, and cyclin-dependent kinases. kinases Regulatory GTPases ras protein Transcription factors myc, myb

1. Growth factors sis gene Homologous with B chain of PDGF int-2, hst Homologous with fibroblast growth factor ( FGF 2. Growth factor receptors C-erb-B EGFR C-fms Colony stimulating factor receptor CSF-1 bit PDGFR ros insulin receptor mas angiotonin AGT receptor or receptor TPK

3. Non-receptor tyrosine protein kinases src abl 4. GTP binding protein H-ras K-ras N-ras ( 21KD- small G protein ) 5. Intranuclear DNA binding protein Fos-Jun transcription factor AP-1 TRE TPA reaction element CREB-JunCRE C-myc basic amio acid single/double strand DNA Rel NF-B associated protein

2 Activation mechanisms of pro-onc 1 1 Insertion of regulatory sequence ( promoter, enhancer) ALV with v-onc induces B cell lymphoma Experiment of Leder group trans-oncogene animal model LTR of mouse mammary tumor virus ( MTV ) C-myc MTV/myc 2. Gene mutation Gene mutation within key modulin of cell carcinoma of bladder T24 cell C ras H T35 --- Val 12 normal G35 --- Gly 12 3 3 Gene rearrangement Burkitt lymphoma 8q24(c-myc) 14q32 close to immune globulin There is a strong enhancer in heavy chain region of immune globulin, and causes overexpression of C-myc. Philadelphia chromosome ( balanced translocation ) 9q34 22q11 C-abl interchanging with bcr

LTR inserting near c-myc inserting

TCR T cell receptor Gene translocation Burkitt lymphoma

fusion gene of bcr-abl in CML Gene translocation Philadelphia chromosome Chromosome 22-bcr geneChromosome 9-c-abl gene Breakpoint in ALL Breakpoint in CML(bcr=5.8kb) Breakpoint in AML and CML 160KD of Bcr protein140KD of Abl protein fusion gene of bcr-abl in ALL 18.5KD of fusion protein 210KD of fusion protein ALL: Acute lymphoblastic leukemia ; CML: Chronic myelogenous leukemia

4. Gene amplification Homogeneously staining regions, HSR Homogeneously staining regions except normal staining regions Double minutes, DM Punctiform pseudochromosome 5 Gene couple Under certain conditions, some pro-oncs are sequentially activated due to activation of other pro-oncs. Products of oncogene promoting immortalization of cell locate in nucleus. C-myc Products of oncogene promoting proliferation of cell locate in cytoplasm. C-ras 6 Elimination of intergenic suppression Special fragment regulating gene expression No.1 exon of C-myc gene dose not encode protein, and may have inhibitory action,if loss, causes activation of c-myc. Methylation of DNA increases stability of double helix.

3 Function of oncogene Immortalization of cell Blocking cell differentiation retrieving cell apoptosis, often locating in nucleus. Transformation of cell Decreasing dependence of cell proliferation on growth factors often locating in cytoplasm or cell membrane.

Extracellular signal: EGF PDGF and so on. GRB 2 SOS Raf Regulating activity of other proteins MAPKK MAPK P P P fos jun myc Regulating gene expression (cyclinD) P P Ras- GTP MAPKKK MEK ERK PI3K PIP3 Akt Anti-apoptosis promoting proliferation PTEN - Two Main signal transduction pathway associated with growth Having mutation in 90% malignant tumor Ovarian cancer Pate cancer Breast cancer Cell membrane Neuclus

Three Tumor suppressor gene Concept A tumor suppressor gene, or anti-oncogene, is a gene that protects a cell from one step on the path to cancer. Tumor-suppressor genes, or more precisely, the proteins for which they code, either have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both. 1. Mostly are recessive genes and not easy to be discovered. 2. Theoretically, the number of oncogenes matches that of tumor suppressor genes. 3. The position of oncogene and tumor suppressor gene may be equally important.

Discovery of tumor suppressor gene: 1. Analysis of cell genetics 70s KundsonPredisposing gene of retinal glioblastoma Retinoblastoma Rb deletion Existence of tumor suppressor genes 2. Experiment of cell hybridization 70s HarrisHybridoma experiment Proving the exsitence of tumor suppressor gene Cognitive process oncoprotein oncogene tumor suppressor gene

Experiment of cell hybridization Normal cell Tumor cell Nontumorous hybrid cell Cell fusion Tumor cell 1 Tumor cell 2 Cell fusion Nontumorous hybrid cell Normal cell Loss of some genes Tumor cell

p53 Predisposing gene of many kinds of tumors P53 So far, the most relevant gene with human tumor. Mutation form 1. Complete loss of p53 gene 2. p53 gene mutation : Two allelic inactivation of p53 One allele for p53 gene inactivation,molecules of p53 with mutation dimerise with wild-type p53 and prevent them from executing functions.

1. Gene localization 17p13.1 2. Gene structure 11 exons No.1 does not code for protein. 33. Protein structure 53kDa ( 7 domains Th 20min ) p53 Acid regionCore regionBasic acid N- -C Easy to be hydrolyzed by protease Phosphorylation site associated with protein stabibility. DNA binding region Important functional position Homo-oligomerisation ( tetramerization ) domain:many modification sites associated with regulating DNA binding activity.

p53 pathway Activation form stability Transcription activity

Mechanism of Growth arrest induced by P53 P53 protein Unwinding enzyme Replication facor A P21protein Holding the cell cycle at G 1 phase Cytocidal DNA damage P21gene P53 protein Replication failure Successful repair suppression

Retinoblastoma Rb 1. Gene localization 13q14 2. Gene structure 27 exons. 3. Protein structure 105kDa P105RB 4. Protein function DNA binding protein binding E2F at phase G0 and G1; Inhibiting transcription activity of E2F; Inhibiting expression of many oncoproteins; Regulating cell cycle. 5. Activity regulation: Phosphorylation decreasing its activity; Dephosphorylation activation. 6. Regulatory factor: Cyclin-CDK Oligomerisation region Binding region of E2F and viral oncoprotein DNA binding region

Mechanism of action of Rb gene G 0, G 1 phase Rb protein E-2F S phase E-2F DNA mRNA DNA P Rb protein

cyclinE A and so on

Kundson Tow-hit hypothesis An inherited, germ-line mutation in a tumor suppressor gene (Rb) would only cause cancer if another mutation event occurred later in the organismss life, inactivating the other allele of that tumor suppressor gene. * It shows for the first time that disease of dominant heredity in family has recessive mutant tumor suppressors alleles. * Mutation form of Rb: Gene deletion and gene mutation; Also associated with osteogenic sarcoma, prostatic carcinoma, small cell lung cancer and so on.

Regulatory of RB and P53 on cell cycle

Function of tumor suppressor gene Inducing terminal differentiation Triggering aging, inducing programed cell death, maintaining gene stabilization Regulating cell growth negative signal transduction Inhibiting activity of protease Changing activity of DNA methylase. Regulating histocompatibility antigen Regulating angiogenesis Promoting interconnection between cells

Inactivation of anti-onc and tumorigenesis 1. gene deletion: A pair of Rb alleles are missing or inactivated before onset 2. gene mutation (1) Loss of Single or both p53 sites reduces concentration of tetramers. (2) Nonsense mutation of P53 caused interruption of translation (3) Loss of acid dmoain in C-terminal affects formation of tetramers; (4) The most common missense mutation: wild-type and mutant to form a more stable tetramer, the loss of normal function. 3. Excessive phosphorylation : 4. Binding with oncoprotein Rb--SV40T antigen adenovirus E1A protein Human papilloma virus E7 protein. P53--SV40T antigen adenovirusE1B protein Human papilloma virusE6 protein. Cause viral DNA replication protein to lose activity. Cause Rb and P53 to lose negative regulatory function of DNA replication.

Other anti-oncs participating in regulating signal transduction APC SMAD4/DPC4 PTEN ( Phosphatase and tensin homolog ) PTEN PTCH ( Patched homolog ) Functions Regulating signal transduction associated with embryonic development and cellular proliferation.

APC ( Adenomatosis polyposis coli ) binding - catenins in the cytoplasm and targeting -catenin for ubuiquitination and degradation by cellular proteosomes ( Wnt/wingless pathway ). SMAD4/DPC4:( Mothers against decapentaplegic homolog 4 ) It belongs to the proteins which modulate members of the TGF protein superfamily. It acts as a tumor suppressor that functions in the regulation of the TGF- signal transduction pathway, which negatively regulates growth of epithelial cells and the extracellular matrix (ECM).

Related genes with DNA reparation MSH2 MLH1 BRCA1 BRCA2 and so on: DNA reparation MSH2 MLH1: DNA mismatch repair

Four Regulation of cells is realized by the coordination of multiple genes Coordination of pro-onc and anti-onc affects cellular proliferation and apoptosis. Malignant cell proliferation is a breach of the normal regulation of cell growth caused by signal transduction.

Five Other diseases associated with cellular proliferation and apoptosis Deficient apoptosis of cells leading to autoimmune disease Excessive apoptosis of cells leading to neural degenerative disease

Six Growth Factor and Receptor 1 Growth Factor 1.Concept A growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. Usually it is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes. 2. characteristics Microamount active material produced by living cell, not nutritional ingredient. Is commonly a polypeptide of 5-80kD, vulnerable to the effects of variability of physical and chemical factors. Mediates biological effects by binding to specific receptors on the surface of their target cells. Has bidirectional biological effects.

Bone morphogenetic proteins (BMPs) Epidermal growth factor (EGF) Erythropoietin (EPO) Fibroblast growth factor (FGF) Granulocyte-colony stimulating factor (G-CSF) Granulocyte-macrophage colony stimulating factor (GM-CSF) Growth differentiation factor-9 (GDF9) Hepatocyte growth factor (HGF) (HDGF) Insulin-like growth factor (IGF) Myostatin (GDF-8) Nerve growth factor (NGF) and other neurotrophins Platelet-derived growth factor (PDGF) Thrombopoietin (TPO) Transforming growth factor alpha(TGF-) Transforming growth factor beta (TGF-) Vascular endothelial growth factor (VEGF) Individual growth factor proteins tend to occur as members of larger families of structurally and evolutionarily related proteins. There are many families which are listed below Diversity Origin Target cells Biological effects

2 Growth Factor Receptors 1.Concept Locat on the cell surface or sub-cells, the protein can specifically recognize and bind its ligand growth factors, and transduct growth factor signals into intracellular, thus activat a series of biological reactions. 2. Construction features N- end rich in Cys binding ligand participating in transducting signals Transmembrane domain rich in hydrophobic amino acid anchoring ligand be fluid C- end key position of transducting singals

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Diseases assotiated with abnomal signal transduction and cellular proliferation and apoptosis