Foundations 2 Week 3

Chromatin regulator

"Chromatin Regulators" is generic term for variety of co-regulatory proteins that interact with Rb protein and facilitate conformation changes needed to release its negative control of E2F transcription factor activity

Wound healing

1. Hemostasis 2. Inflammatory phase 3. Proliferative phase 4. Maturation phase

Hyaluranon

25,000 subunits Made directly outside the cell Most common No SO3 Joint fluid, wound healing, development Source of cell migration Degraded by hyaluronidase Hyaluronan is thought to have a role in resisting compressive forces in tissues and joints. It is also important as a space filler during embryonic development, where it can be used to force a change in the shape of a structure, as a small quantity expands with water to occupy a large volume

Oncogene

A gene, which upon mutation, produces a protein that is over expressed or hyperactive resulting in excessive cell proliferation is defined as an oncogene. Oncogenes are typically components of signal transduction pathways, and over 100 oncogenes have been identified. The normal functional gene counterpart is known as a proto-oncogene. Normal diploid cells have two copies of most genes. Generally, a mutation in only one copy of a proto-oncogene is sufficient to disrupt growth regulation. Therefore oncogenic mutations are thought to be dominant, and tumorigenesis results from the gain of a function in the mutated oncogene. Retroviruses induce cellular transformation as a result of the location within the host genome of the proviral DNA In contrast to proto-oncogenes, usually both copies of a tumor-suppressor gene must be mutated or inactivated for an effect on growth regulation to become apparent. Retinoblastoma (retinal tumor)

Activating Cdk

Allosteric effector is cyclin. CAK is general. Sometimes may need to stockpile product. Checkpoint controls: Modulation of Cdk activity by inhibitory phosphorylation (M-Cdk) or by an inhibitory protein called CKI (G1/S-Cdk)

Sister chromatid separation

Anaphase-promoting complex: ubiquitin ligase might be activated by M-Cdk (although there is a lag) cohesin complex keeps chromatids bound at the centromere at end of metaphase, APC targets securin that inhibits the protease separase Separase targets cohesin complex

Proteoglycans

Are molecule consisting of one or more glycosaminoglycan (GAG) chains attached to a core protein. Proteoglycans are usually easily distinguished from other glycoproteins by the nature, quantity, and arrangement of their sugar side chains. By definition, at least one of the sugar side chains of a proteoglycan must be a GAG The proteoglycan aggrecan: a major component of cartilage, has a mass of about 3 × 10^6 daltons with over 100 GAG chains. largest The proteoglycan decorin: is secreted by fibroblasts and has a single GAG chain. smallest

Morphogenesis

Axis development Craniocaudal Anteroposterior Laterality Asymmetry development determined by clusters of families of transcription factors

Biochemical approach for cell cycle components

Bigger cells such as those from Xenopus frog provide protein extracts for VALIDATING existence of cell cycle regulatory proteins.

Cell cycle control

CHECKPOINTS to allow cell cycle movement in response to PROGRESSION FACTORS Checkpoints also allow for accumulation of microenvironmental signals such as growth factors indicating a general need for cell cycle progression in the tissue bed Operate by negative signals designed to stop the action if something not quite right Mutations in checkpoints do not stop the cell cycle. Losing negative control causes cancer

E2F-independent mechanism for RB-induced cell cycle arrest

Captures S phase kinase-associated protein 2 (SKP2) that adds ubiquitin groups to the p27 cyclin-kinase inhibitor (CKI) needed for p27 turnover. Turnover of p27 CKI needed for G1 progression (panel a) Two models possible: RB binds and inactivates SKP2 (panel b) OR binds both SKP2 and APC thus targeting ubiquitin groups to SKP2 which then becomes degraded (panel c) Bottom Line: Rb can prevent cell cycle progression from 2 vantage points: (1) sequestering E2F needed for G1 CDK production and (2) stabilizing available cyclin-kinase inhibitors (p27)

Tumors and VEGF

Cells monitor O2 tension. Under hypoxic conditions signals are released to enhance production and release of angiogenesis factors such as vascular endothelial growth factor (VEGF). VEGF binds to receptors on the surface of endothelial cells stimulating their proliferation. Endothelial cells also migrate towards areas of higher VEGF concentration. Therefore, VEGF is defined as an angiogenic factor Solid tumors can only reach a certain size without a vascualr supply

Limb bud development

Classic example of morphogen gradient 3-dimensional patterning Proximodistal Anteroposterior Dorsoventral Cartilage and bone differentiation Programmed cell death Regulated by complex network of signal molecules AER = apical ectodermal ridge ZPA = zone of polarizing activity PZ = progress zone AER produces fibroblast growth factors (FGF), needed for proximodistal growth ZPA produces Sonic hedgehog (SHH), regulates patterning in anteroposterior axis Limb identity (upper v lower) determined by TBX proteins All are interdependent and have complex feedback loops Achondroplasia GOF of FGFR3 (FGF receptor 3) Defective differentiation of chondrocytes leads to growth retardation of longitudinal bones Proper balance of FGF needed for proximodistal growth of limb bud

Fate of sister chromatids

Cohesins (interstrand) are multisubunit proteins that are deposited along the length of replicated chromatin to link sisters together Condensins (intrastrand) are proteins that cause replicated chromatin to condense about 50-fold to produce visible chromosomes. Condensin mediates chromatin compaction during prometaphase/metaphase M-Cdk activates condensins which use ATP to drive coiling process Active Rb may assist with the maintenance of chromatin structure during G1 and M phase

Heterotypic interaction

Communication between different cell types (including the tumor cells) Epithelium is an avascular tissue. Epithelial tissues give rise to carcinomas (~80% of all tumors). 90% or more of the cells in a carcinoma are normal tissue stromal cells: Fibroblasts, endothelial cells, pericytes,smooth muscle, adipocytes, macrophages, lymphocytes, etc.

Components of an imprinted gene cluster

Components of an imprinted gene cluster. The maternal (top) and paternal (bottom) alleles of an imprinted gene cluster are depicted. Imprinted gene clusters contain maternally expressed (pink-filled boxes), paternally expressed (blue-filled boxes), and biallelically expressed genes (i.e., nonimprinted genes, green-filled boxes). These nonimprinted genes can be found in the middle of a cluster surrounded by imprinted genes. The ICR (yellow) controls imprinting of multiple genes in the region; deletion of this differentially methylated element results in loss of imprinting of the linked genes. Many imprinted clusters also contain additional DMRs (orange) that acquire DNA methylation after the preimplantation stage. Not depicted here are long ncRNAs and insulators, which have an essential role in imprinted clusters.

Capillary categories

Continuous is major type. Focus on low. Fenestrated- punctured sites, not free floating cytoplasm. Discontinuos- large gaps between cells

Cyclins

Cyclins undergo cycles of synthesis and degradation in each cell cycle. Cyclins are rate-limiting proteins that bind kinases (Cdks) that usually are ubiquitous in the simplest cell cycle schemes. Disposable

DNA methylation

Cytosine-->5-methylcytosine Doesn't affect DNA binding, but influences how other entities can bind to the DNA CpG islands are primary target Located in 5' ends (promoter or exon 1) of genes Found in or near 70% of human genes Hypermethylation correlates with transcriptional repression Hypermethylation can persist from the parental germ line into the zygote (parent of origin effect or "imprinting") Near genes of regulation. So following genes can be shut down

S phase intracellular controls

Cytosol from S phase cells can trigger DNA replication in the G1 nucleus, but not a G2 nucleus The clock must therefore be reset after S phase to allow future rounds of DNA replication. Once cells reach G2, they ignore the S phase signal Clock is reset by M phase

Disruption

Destruction of normally developing tissue Vascular disruption External factor Recurrence risk <<1% Sac gets disrupted and interferes with development

HOX genes

Developmental transcription factors Homeotic box genes Similar to genes in Drosophila responsible for body segmentation Homeobox = three linked α helices (helix-turn-helix family of DNA-binding proteins) 39 HOX genes in humans Four clusters on four chromosomes Temporospatial arrangement Two exons in each, second exon has homeobox Same HOX protein can be a repressor at one gene and an activator at another Lowest frequency of repeats in the entire human genome Need to conserve precise arrangement of genes and regulatory elements within gene clusters Many ncRNAs (~230 in humans) May be transcribed from one cluster and act on another 1 first, 13 last HOX gene overlap determines vertebral development. Expression continues into adulthood Maintain regionally specific expression

Differentially methylated regions (DMRs)

Differ between maternal and paternal loci Cis-acting mechanism Can act over short or long distances Somatic differentiation Sometimes tissue specific Depend on the presence of a germline DMR Germline DMRs Oogenesis: found at promoters of protein-coding or ncRNA genes Spermatogenesis: found in intergenic regions Also known as "Imprinting Control Region"

Histone modification

Each nucleosome composed of octamer of histones Needed for compaction of chromatin. Histone tails stick out through DNA strands. Post-translational modification on the tails Acetylation (-CH2CH3) Acetylation of lysine residues in the tail of histone H3 by histone acetyltransferase enzymes (HATs) May change altered electric charge to separate DNA from histones May create a binding site for chromatin modifying enzymes and basal transcription machinery Regardless, correlates with ability to be transcribed Deacetylation by histone deacetylase (HDAC)- Condenses chromatin structure, decreased gene transcription Methylation (-CH3) Methylation may allow binding of a transcriptional repressor protein Constitutively transcriptionally silent chromatin (constitutive heterochromatin) Also ubiquitylation, phosphorylation, sumoylation Different combinations of modifications may have different actions

Epidermal Growth Factor (EGF) Family

Epidermal growth factor (EGF) family of mitogens comprises several members, including EGF, transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), amphiregulin, epiregulin, betacellulin, neuregulins, the recently discovered epigen. All these growth factors exert their functions by binding to four different high-affinity receptors, EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4 EGF has been implicated in keratinocyte migration, fibroblast function and the formation of granulation tissue during wound healing. HB-EGF shedding can be inhibited by compound OSU8-1. Interestingly, the application of this compound to full-thickness mouse wounds caused a strong retardation of reepithelialization as a result of impaired keratinocyte migration suggesting a role of HB-EGF in wound re-epithelialization.

Matrix Metalloproteinases

Epidermis- collagenase (MMP-1), stromelysin-1,2 (MMP-3,10), plasminogen activator (urokinase type) Mesenchyme- collagenase (MMP-1), stromelysin 1 (MMP-3), 72kDa gelatinase (MMP-2), 92kDa gelatinase (MMP-9), plasminogen activator (uPA) MMPs secreted as zymogens Complex activation pathways Counteraction by Tissue Inhibitors of Metalloproteinases (TIMPs) Injury exposes epithelia to a new ECM environment - collagen I/fibrin/fibronectin Migration on to these substrates requires Reprogramming of matrix receptors (integrins) Loss of intercellular contacts Activation of MMP pathways MMP-13 Expression is Elevated in the Cutaneous Wound Higher expression observed in Free electron laser wounds up to 42 days than scalpel injury Granulocyte Proteinases: Serine proteinases- elastase, cathepsin G, proteinase 3, azurocidin, neutrophil collagenase (MMP-8) Macrophage proteinases: collagenase (MMP-1), metalloelastase (MMP-12) Modulators of proteinase activity: Serpins, Maspins, TIMP's, SLPI, a-2 macroglobulin, PAI-1

Mir-200

Example of disruption of normal epigenetics CpG island hypermethylation of miR200 family Lack of miR200 transcription Permits expression of several transcriptional repressors Allows repression of E-cadherin gene normally responsible for cell-cell adhesion Permits epithelial-to-mesenchymal transition of cancer and allows cancer to invade

Deformation

External factors exerting mechanical force Malformation caused by Potter Sequence

Detection of mosaicism

Extra band- because of 3 copies. AAA, AAB, ABB, BBB

Fibroblast Growth Factor (FGF)

FGFs comprise a growing family of structurally related polypeptide growth factors, currently consisting of 22 members. They transduce their signals through four high-affinity transmembrane protein tyrosine kinases, FGF receptors 1-4 (FGFR1-4), which bind the different FGFs with different affinities. Additional complexity is achieved by alternative splicing in the extracellular domains of FGFR1-3, which dramatically affects their ligand binding specificities. FGF1, however, binds to all known receptors, and FGF7 specifically interacts with a splice variant of FGFR2, designated FGFR2IIIb. A characteristic feature of FGFs is their interaction with heparin or heparan sulfate proteoglycans, which stabilizes FGFs to thermal denaturation and proteolysis, and which strongly limits their diffusibility. Most importantly, the interaction with heparin or heparan sulfate proteoglycans is essential for the activation of the signaling receptors. FGF family are known to stimulate proliferation of various cells of mesodermal, ectodermal, and also endodermal origin The only exception is FGF7 ( aka. keratinocyte growth factor, KGF), which seems to be specific for epithelial cells, at least in the adult organism. FGFs also regulate migration and differentiation of their target cells, and some FGFs have been shown to be cyto-protective and to support cell survival under stress conditions.

Epigenetics in early embryo

Fertilization Oocyte and sperm have CpG methylation Sperm immediately demethylates Oocyte slower First DNA replication Remethylation begins to appear

E2F transcription factors

Five members: E2F1-5 Trans-activate cell cycle-associated gene Form heteromeric complexes needed for induction of gene transcription Regulated by direct interaction with pocket proteins, e.g. Retinoblastoma protein

Escape from G1 phase

G1-Cdk activation is key to ready cells for S phase and can be linked to growth factor receptor-activated kinases (MAP kinases such as ras). Hct1-APC does not block G- or G1/S-cyclins. In fact, G1/S-Cdk and S-Cdk inactivate Hct1-APC. Removal of Hct1-APC sets the stage for eventual need for M-cyclin and M-Cdk. G1-Cdk activates retinoblastoma protein (Rb) reducing its lock-up of E2F, a powerful transcriptional activator of genes needed for escape from G1 into S phase such as G1/S-cyclin

Angiogenesis

Growth of New Blood Vessels from preexisting blood vessels Vasculature plays an important functional role in: 1) embryonic development; 2) implantation of the placenta; 3) wound healing; 4) disease states such as diabetic retinopathy, psoriasis, and rheumatoid arthritis; and 5) tumorigenesis. Tumors do not invade normal tissue and capture existing blood supply, rather they recruit endothelial cells to form new capillaries. Stimulated by hypoxia Oncogenes govern the switch Numerous factors control & inhibit it Formation of new blood vessel from: 1) Preexisting vessels or 2)Endothelial progenitor cells from the bone marrow.

Imprinting pedigree

Here, the mutation is in a maternally expressed imprinted gene, and therefore, when the father transmits the mutation, offspring are unaffected because the paternally inherited allele is normally repressed. Half of his offspring will be carriers (as in generation II) - and follows Mendelian rules but the expression of the phenotype is regulated by the imprinting status of the gene. The female carriers will transmit the mutation in the active, maternally inherited allele to 50% of their children, who will be affected, as is clearly evident in generation III. Even though the affected individuals have a completely shaded in square, they still only have a single mutated copy of the gene.

Hyperplasia and dysplasia

Hyperplasia results when cells of a tissue are stimulated to undergo mitotic divisions, thereby increasing the number of cells. Dysplasia generally refers to an expansion of immature cells with a parallel reduction in mature cells. Dysplasia is often an early stage in the neoplastic process.

Retinoblastoma (Rb) protein

Hypophosphorylated form (active form of protein) Binds transcription factors of the E2F family Hyperphosphorylated form (inactive form of protein) Releases E2F transcription factors Regulates passage through G1/S restriction point of cell cycle Retinoblastoma results from inactivation of both copies of the Rb gene: Rb is a tumor suppressor gene. Related pocket proteins are p107 and p130. Many cancers find a way to impair Rb structure; or alter expression of kinases (Cdks) or cyclins or Cdk inhibitors (p27) that impact Rb structure/function

Cell biology approach for cell cycle components

Imaging replicated DNA in single cells or cell populations provides KINETIC & SPATIAL information about S phase activity

Effect measures in cohort studies

Incidence in exposed = a / a+b Incidence in unexposed = c / c+d Relative risk = (a / a+b)/(c / c+d)

Therapeutic programming: Reprogrammed mature cells

Induced pluripotent stem cells (iPS) Adult somatic cell and nucleus No ovum needed Genetically identical to donor Inducing a forced expression of certain genes Requires transfection with stem-cell specific genes - Oct4, Sox2, cMyc, Klf4 Avoids ethical issue of embryo origin Risks Propensity to form tumors (teratomas) All transfected genes linked with carcinogenesis Altered CpG methylation patterns and influences on cellular differentiation

Uniparental Disomy

Inheritance of both homologues of a chromosome or region of a chromosome from the same parent; no copies from the other parent Whole chromosome UPD results when trisomy of fertilized ovum is rescued by loss of the 3rd chromosome Parent-of-origin genes affected Two active genes --> overexpression, + harmful effect Two inactive genes --> same as deleting gene Can tolerate over expression better than under expression A) no heterozygosity- single chromosome duplicated. Monosomy rescue B) the homozygous regions indicate uniparental isodisomy and the heterozygous regions indicate uniparental heterodisomy. In this patient, the two different chromosomes from one parent must have been present in the resulting trisomic zygote, which is resolved by the postzygotic loss of the one chromosome from the other parent. Trisomy rescue

ncRNA as target for cancer therapy

Inhibit miRNA function if upregulated miRNAs regulate through base pairing Use antisense oligonucleotides to inhibit miRNA Restore miRNA function if downregulated DNA demethylating agents Histone deacetylase inhibitors Nanotechnology to distribute miRNAs

Macromolecules in the ECM

Insoluble components- (fibrous proteins): Collagen Elastic fibers Fibronectin Laminin Soluble components-(polysaccharide chains) - Proteoglycans Hyaluronan Adhesive glycoproteins

Malformations

Isolated or part of a syndrome with other defects Flawed process of formation from the beginning Recurrence risk to other family members Inheritance of syndrome, or Isolated - multifactorial, 3-5% Major: needs surgery Minor: live with it Isolated caused by multifactorial pattern. Genetic polymorphisms and smoking is high risk for cleft palate for example

Laminin

Laminin-1 (classical laminin) is a large, flexible protein composed of three very long polypeptide chains (α, β, and γ) Laminin-322 is deposited as a part of a provisional matrix during wound healing. Reduced laminin-322 levels has been associated with poor keratinocyte migration The keratinocyte's primary anchoring contacts are hemidesmosomes, which bind to laminin in the basal lamina by way of a6b4 integrins and have intracellular links with the keratin cytoskeletal network. During wound healing, laminin-5 is expressed by keratinocytes at the leading edge of the dermal-epidermal junction. The epidermal growth factor-like repeats in laminin are examples of matrikines. Matrikines are peptides liberated by partial proteolysis of extracellular matrix macromolecules.

DNA damage checkpoints

Late G1: prevents S phase entry by stimulating p53 that activates transcription of the CKI called p21 that inhibits both G1/S-Cdk and S-Cdk Late G2: prevents M phase entry by stimulating kinases that inactivate the Cdc25 phosphatase needed for M-Cdk activation

M phase intracellular controls

M cyclin levels increase during G2/M Phase M-Cdk is held in latent state by Wee1 kinase and then activated by cdc25 phosphatase There is an activating cascade at work at this point involving a cdc25 activator (Polo kinase) and M-Cdk itself. DNA replication-fork inhibitor of cdc25 now gone. M-Cdk activates its own activator (cdc25) and inhibits its own inhibitor (Wee1)

G1 Phase

M-Cdk inactivation at end of M phase facilitated by the Cdc20-APC complex. G1 phase is a time of Cdk inactivity To assure this, a Hct1-APC complex is formed in late M phase after the Cdc20-APC complex has started destroying M-cyclin. It assures full destruction of remaining M-cyclin as well as Cdc20 removal Also, CKIs (p27, p21) accumulate that block any G1/S-Cdk to prevent premature entry into S phase

Anomalies and meanings

Malformation - structural defect of organ resulting from the abnormal formation of tissues. Major vs minor Three or more minor malformations raises concern Syndrome - multiple anomalies with a single basic cause which occur independently rather than sequentially Disruption - structural defect resulting from tissue damage and breakdown of otherwise normal structures Deformation - an abnormality in form or position of a body part caused by mechanical force (constraint) or secondary effects from a functional abnormality in the fetus Sequence - a structural defect or mechanical factor which leads to a sequence of secondary effects (if-->then)

Retinoic Acid

Metabolite of vitamin A Mediates the functions of vitamin A required for growth and develop Binds to a receptor which binds directly to specific DNA regions Regions near HOX genes Isotretinoin (13-cis-retinoic acid) Oral medication for cystic acne and severe skin disorders Low affinity for retinoic acid receptors but may be converted intracellularly to metabolites that are agonists FDA Category X contraindicated in pregnancy because of teratogenicity Hydrocephaly Microcephaly Intellectual disabilities Ear and eye abnormalities Cleft palate and other facial abnormalities Heart defects

Genetic Evidence for Rb/SKP2 Crosstalk Independent from E2F

Mice heterozygous for Rb mutations (Rb+/-) normally have pituitary or thyroid cancer When crossed with SKP2-deficient mice, cancer incidence was reduced E2F activity in these mice indeed was active due to lack of binding to Rb. Paradox: high E2F usually means CDK expression & cell cycle progression However, cell proliferation was kept in check in the absence of SKP2 by continued availability of p27 CKI Excess p27 also enhanced apoptosis in pituitary

Epigenetic mechanism for imprinting

Must be able to influence transcription Must be heritable in somatic cell lineages The parental origin of a gene locus is faithfully passed on to daughter cells during mitosis Likely to be initiated on paternally and maternally inherited chromosomes at a time when they are not in the same nucleus During gametogeneis or immediately after fertilization Must be erased in the germ line Appropriate parent-of-origin identity of a gene locus can be established in the games for the next generation Only one allele expressed in at least one body tissue Occur in clusters- Other imprinted genes Imprinting control center with parental-specific DNA methylation At least one ncRNA Alleles inherited through generations will change methylation and activity pattern depending on sex of transmitting parent Frequently involves CpG methylation

Extracellular Matrix

Network of proteins and carbohydrates that binds cells together Supports and surrounds cells Regulates cells activities Lattice for cell movement This tissue contains a variety of cells and extracellular matrix components. The predominant cell type is the fibroblast, which secretes abundant extracellular matrix

Angiogenic switch

Normal conditions prevent cells from triggering angiogenesis. As tumors progress they gain the capacity to promote angiogenesis Thus, overcoming the normal inhibition of angiogenesis is a step or "switch" in tumor progression. Only a small number of hyperplastic islets gain capacity to progress into tumorsGain of angiogenic capacity ("switch") Angiogenic capacity is gained prior to penetration of the basement membrane Once the Tumor Becomes Invasive (Malignant) it Gains Direct Access to Stromal Cells

Cohort study

Observational study Follow group of individuals forward through time to capture outcome To examine incidence To look at the natural history of disease To assess the temporality of an exposure/disease relation (when a RCT is not feasible) The ONLY way other than a RCT to ensure that exposure precedes disease Expensive, not good for rare or latent diseases

X chromosome inactivation

Occurs in all mammals Selective epigenetic inactivation of alleles on one X Require presence of 2 or more Xs Random selection of X in each cell of embryonic inner cell mass Descendants of each cell will have the same X inactivated from that time on All females are somatic mosaics for X gene activity Transcription and cis- limited spreading of a long ncRNA ncRNA coats X chromosome DNA over very long distances and inactivates it Female carriers of x-linked recessive will have phenotype determined by distribution of mosaicism of X eg. DMD and Hemophilia A

Embryologic development

Only bottom 3 are parts of the actual fetus Cellular differentiation produces specific cell types Totipotency - first 2-4 cells Have the potential to develop into a complete organism 16 cell stage, differentiation has begun Embryo vs amnion vs chorion Pluripotent - cells of inner cell mass (embryo) can no longer produce the trophoblast (placenta) Multipotent - germ layer cells (ectoderm, mesoderm, endoderm) cannot produce cells of another layer Unipotent - can only produce a single type of differentiated cell Stem cells - precursor cell capable of self-renewal as a continual source of differentiated cells Classified by potency

Platelet-Derived Growth Factor (PDGF)

PDGFs comprise a family of homo- or heterodimeric growth factors, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD PDGF exert their functions by binding to three different transmembrane tyrosine kinase receptors, which are homo- or heterodimers of an α- and a β-chain Upon injury, PDGF is released in large amounts from degranulating platelets at wound site The expression patterns of PDGF and PDGFR at wound site suggest a paracrine mechanism of action, the ligands are predominantly expressed in the epidermis, whereas the receptors are found in the dermis and the granulation tissue. The expression of PDGFs and their receptors are decreased in wounds of healing-impaired genetically diabetic db/db mice and glucocorticoid-treated mice Augmented PDGF production might be involved in the pathogenesis of hypertrophic scars and keloids as suggested by the potent effect of PDGF on fibroblast proliferation and extracellular matrix production.

Parent-of-origin Effects: Genomic Imprinting

Parent-of-origin determines whether some genes are expressed or not ~100 autosomal genes are imprinted Only one allele expressed in these gene pairs Has nothing to do with sex-linked inheritance (X and Y chromosomes) Imprinted gene = inactivated gene Inheritance process independent of classical Mendelian inheritance Epigenetic changes established in the germline and maintained throughout mitotic divisions Normal imprinting: Growth of embryo, Placental function, Stem cell programming, Neurobehavioral processes, Memory and synaptic functions Disrupted imprinting: Carcinogenesis, Diseases showing abnormal physical growth and neurobehavioral development

Imprinting vs mendelian inheritance

Paternal gene not expressed I-1: Dark blue allele expressed came from mother I-2: White allele expressed came from mother II-1: White allele expressed came from mother Why is the light blue gene active in III-1 when it was inactive in II-1? Reset when inherited from mother Why is the white gene inactive in III-1 when it was active in II-2? Reset when inherited from father

Imprinting and dominant inheritance

Paternally expressed form of an imprinted gene Loss-of-function mutation in one allele Only seen in "dominant" mutations

Pocket domain

Pocket domain binds viral oncoproteins E1A (adenovirus), T-ag (SV40), E7 (HPV) at LXCXE. E2F binds at A-B junction and RBC. RBN resembles the pocket but the RBC is disordered Disordered regions (black lines) near RBC are cyclin-CDK targets Phosphorylations (~13) cause folding and prevent engagement of E2F Folded conformation is inactive and recycled by phosphatase PP1 Important phosphorylation hit sites that affect Rb structure are located in the disordered regions (gray shaded regions) that flank the ordered pocket and RBN domains. Evolution acts on these to protect essential domains such as RBN & pocket

Growth factors

Polypeptides produced in normal and wounded tissue Stimulate migration, proliferation, differentiation, gene regulation and function Extremely potent act over very small distances and influence cellular function via receptors Autocrine or paracrine

Cdc6 and MCM in S phase control

Pre-replicative complex forms during G1: Cdc6+MCM proteins Once S-Cdk fires off, it helps to destroy Cdc6 (via SCF and ubiquitin) and phosphorylates MCM to cause their nuclear export thus preventing further DNA replication S-Cdk remains high during G2 but eventually is reduced after M phase. M-Cdk also phosphorylates Cdc6 and MCM to prevent DNA replication during M phase. No more pre-RC in G2

Induction

Process by which one part of an embryo causes adjacent tissues to change through diffusion of substances Sequential induction - morphogens released in a series of signals provided by different agents

Genomic instability independent from faulty cell-cycle progression

Rb mutations can lead to defects in loading of condensin along prometaphase chromosomes and cohesin at the centromere Condensin promotes condensation and cohesin holds the sister chromatids together Unregulated E2F causes high expression of MAD2 that prevents timely attachment of chromosomes to spindle microtubules (spindle checkpoint) Together, lack of Rb and excess E2F activity results in tangled chromosomes and aneuploidy Mad2 marks a kinetochore that has failed to attract a microtubule. The laggard chromosome sends a negative signal that prevents APC-Cdc20 from activating separase. Rb mutants may cause excessive transcription of Mad2 via E2F and disallow normal metaphase

Angiogenic signaling

Real blood vessel breaks and migrates toward tumor cell. MMP paves the way Initially the tumor recruits local endothelial cells, but it has been shown that as the tumor matures it can recruit precursor cells from the bone marrow to the tumor site even prior to metastasis of the tumor.

PDGF in wound therapy

Regranex: Becaplermin Topical gel recombinant human PDGF for topical administration The ONLY currently available commercial product based on growth factor therapy for chronic wounds like ulcers Can cause malignancies Platelet rich plasma (PRP)- Concentration of autologous platelets greater than the peripheral blood concentration suspended in a solution of autologous plasma. heals soft tissue and used for bone re-generization Synonyms: Platelet Concentrates (PC) Plasma Very Rich in Platelets (PVRP)

Control of proteolysis by SFC

Reserves of cyclin:Cdk complexes also can be activated & deployed or destroyed by ubiquitin-dependent proteolysis Ubiquitin ligase: SCF helps degrade p27 CKI that restrict G1/S and S phase progression

Collagen

Resists tensile forces, major insoluble fibrous protein in ECM and connective tissue, most abundant protein in animals, made by fibroblasts and epithelial cells -Triple alpha helix structure Fibrillar collagen- tendon, cartilage. well known striated fibrils. types I-III, V and XI Sheet forming collagen- Polymerizes into sheets. Forms basal membranes - collagen IV. Collagen VIII - Descemet's membrane of cornea Connecting collagen- Link fibrillar and sheet forming collagens to into networks and connect them to other structures. Collagen VI - short helices interspersed with globular domains. Align collagen I into parallel structures

Cell Cycle

S phase- 50 nucleotides/second. 8-12 hours M phase- 1 hour G1 and G2- 11 to 13 hours Bone marrow progenitors: 12 to 24 hours Intestinal epithelium: 12 to 14 hours Bronchial epithelium: 9 to 10 days Hepatocytes: 150 days Cardiomyocytes: Never, in an unassisted state. Sometimes, the signals never emerge resulting in G0 where cells can persist for years before resuming proliferation, if at all

Spindle attachment checkpoint

Sensor that detects attachment of kinetochores to microtubules Failed attachment generates a negative signal that blocks anaphase Mad2 binds unattached kinetochores and blocks Cdc20-APC induced destruction of securin by sequestering Cdc20 M-Cdk and Polo kinase both release Cdc20 when all kinetochores become attached to the spindle Mad2 marks a kinetochore that has failed to attract a microtubule. The laggard chromosome sends a negative signal that prevents APC-Cdc20 from activating separase

Collagen in wound healing

Serves as structural support control many cellular functions, including cell shape and differentiation, migration, and synthesis of proteins There are a number of different collagen dressings available, which employ a variety of carriers/combining agents such as gels, pastes, polymers, oxidized regenerated cellulose (ORC), and ethylene diamine tetraacetic acid (EDTA). Purified to be non-antigenic A given collagen dressing may contain ingredients, such as alginates and cellulose derivatives that can enhance absorbency, flexibility, and comfort, and help maintain a moist wound environment Proteinase resistant triple helix Cleaved by collagenase (MMP-1. inactive form and active form)

Glycosaminoglycans (GAGs)

Soluble, polysaccharide chains, linked to protein in the form of proteoglycans unbranched polysaccharide chains composed of repeating disaccharide units. They are called GAGs because one of the two sugars in the repeating disaccharide is always an amino sugar which is in most cases sulfated 4 Groups, distinguished according to their sugars, the type of linkage between the sugars, and the number and location of sulfate groups: Hyaluronan chondroitin sulfate and dermatan sulfate heparan sulfate keratan sulfate.

Activation of Smad proteins by transforming growth factor receptors

TGF-β is first produced as an inactive precursor that binds to latency-associated protein (LAP). Upon activation, TGF-β is either sequestered by extracellular binding proteins (decorin, fibromodulin) or it binds to a type III receptor that presents it to the signal-transducing receptors (type II and type I). Upon ligand binding, TGF-β type II receptor recruits and phosphorylates the type I receptor. The latter subsequently binds and phosphorylates Smad2 and Smad3. Phosphorylated Smad2 and Smad3 bind to Smad4 and translocate to the nucleus where they bind to other transcription factors that confer specificity, leading to activation of target genes.

Transforming Growth Factor-β

TGF-β superfamily encompasses a diverse range of proteins, many of which play important roles during development, homeostasis, disease, and repair. The three mammalian TGF-β isoforms (TGF-β1, -β2, and -β3) are synthesized as latent precursors, usually being secreted as a complex with latent TGF-β-binding protein, which is then removed extracellularly via proteolytic cleavage. Active TGF-βs then exert their biological functions via binding to a heteromeric receptor complex, consisting of one type I and one type II receptor, both of which are serine-threonine kinases. In addition, they bind with high affinity to a nonsignaling type III receptor, which functions mainly to present TGF-β to the type II receptor. The three TGF-β isoforms have both distinct and overlapping functions. Mitogenic for fibroblasts but not for other cell types Potent stimulators of ECM proteins and integrins

Genetic approach for cell cycle components

Temperature-sensitive yeast mutants: Cell cycle control genes PREDICTED by G1/S blockade (or blockage at any other boundary). Essential kinases are rate-limiting for cell cycle progression. If non-functional, cell cycling stops at point where needed.

UPD of entire genome (46,XX)

Teratomas are largest tumors Right- can act like malignancy

Mitogens and G1 exit

The MAP kinase c-ras elevates c-myc that activates cyclin D and G1-Cdk as well as SCF ubiquitin ligase that degrades p27 leading to increased G1/S-Cdk G1-Cdk and G1/S-Cdk phosphorylate Rb to release E2F and enhance S phase gene expression

Elastic Fibers

The main component of elastic fibers is elastin, a highly hydrophobic protein (about 750 amino acids long). Like collagen, is unusually rich in proline and glycine but, unlike collagen, is not glycosylated and contains some hydroxy-proline but no hydroxylysine. Soluble tropoelastin (the biosynthetic precursor of elastin) is secreted into the extracellular space and assembled into elastic fibers close to the plasma membrane, generally in cell-surface in foldings. The molecules are joined together by covalent bonds (red) to generate a cross-linked network. In this model, each elastin molecule in the network can expand and contract as a random coil, so that the entire assembly can stretch and recoil like a rubber band. Crosslinked by lysyl oxidase Crosslinking results in incredibly insoluble and durable polymer Cutis laxa (CL), or elastolysis, is a rare, inherited or acquired connective tissue disorder in which the skin becomes inelastic and hangs loosely in folds). Marfan syndrome. Marfan syndrome is a disorder of connective tissue which causes skeletal defects typically recognized in a tall, lanky person. A person with Marfan syndrome may exhibit long limbs and spider-like fingers, chest abnormalities, curvature of the spine and a particular set of facial features including a highly arched palate, and crowded teeth.

Stromal support for tumor survival

The threshold for oxygen diffusion is approximately 0.2 mm. Tumor cells will cluster around blood vessels. If more than 0.2 mm from vessels, tumor cells subject to death from hypoxia

Non-coding RNA (ncRNA)

Translation- tRNA, rRNA snoRNA (small nucleolar), snRNA (small nuclear) Gene expression regulation- microRNA, siRNA (small interfering), piRNA (PiWi), long ncRNA Will not be picked up in exomic sequencing tRNA is the only ncRNA that acts alone microRNA (miRNA) (Mirs) About 2000 discovered in humans Regulate about 60% of human coding genes Each miRNA may regulate up to 200 transcripts Complementary to mRNA in 3'UTRs Causes mRNA to decay, reduces gene product miRNA in turn controlled by CpG island methylation Piwi-interacting RNAs (piRNAs) Transcriptional silencing in spermatogenesis All involved in developmental gene regulation Deregulation of ncRNA leads to immune disorders, cancer, and neurodegenerative diseases

Tumor vs normal capillaries

Tumor capillaries are: 3 times the diameter Disorganized layout Loosely associated pericytes Gaps of several microns between cells Walls up to 10 times more permeable Higher interstitial fluid pressure Poor lymphatic drainage High interstitial pressure in tumor lowers effective penetration of anti-tumor drugs. Increased capillary density within the tumor has a poor prognosis for survival

Multiple Heterotypic Interactions are Involved in Angiogenesis

Tumor cells may attract other stromal cell types, that in turn promote angiogenesis. Tumor cells may release enzymes, matrix metaloproteinases (MMP), that degrade the extracellular matrix and basement membrane. Endothelial cells can produce when activated also releases ECM bound angiogenic factors like VEGF. promotes endothelial cell migration through ECM. allows for direct access of tumor to new vessels. Tumor cells may attract local endothelial cells, but can also attract precursor cells from distant sites like the bone marrow.

Control of proteolysis by APC

Ubiquitin ligase: APC (anaphase promoting complex) destroys M cyclin and terminates M phase

DNA Replication G2 checkpoint

Unfinished replication forks send a negative signal to M-Cdk The negative signal activates a kinase that inhibits the Cdc25 phosphatase and allows Wee1 to keep M-Cdk in inactive state M-Cdk induces assembly of mitotic spindle, chromosome condensation, nuclear envelope breakdown, actin-myosin cytokinesis, and distribution of membranous organelles to daughter cells

Effects of TGF-beta

Upon local hemorrhage, TGF-β is released in large amounts from platelets. In the healing wound, it is produced by leukocytes, macrophages, fibroblasts, and keratinocytes and acts on these cells to stimulate infiltration of inflammatory cells, fibroplasia, matrix deposition, and angiogenesis. In contrast, endogenous TGF-β has been shown to inhibit re-epithelialization. Functions in late stage wound repair TGF-b1 and TGF-b2: Promotes angiogenesis, up-regulates collagen production and inhibits degradation, promotes chemoattraction of inflammatory cells. TGF-b3 (antagonist to TGF-b1 and b2): Has been found in high levels in fetal scarless wound healing and has promoted scarless healing in adults experimentally when TGF-b1 and TGF-b2 are suppressed. avotermin (by Renovo) - a recombinant human transforming growth factor beta 3 for scar treatments. Failed

Angiogenic factors

VEGF (Vascular Endothelial Growth Factor) TGF-beta (Transforming Growth Factor Beta) bFGF (Basic Fibroblast Growth Factor) Interleukin-8 Angiopoietin Angiogenin PDGF (Platelet Derived Growth Factor)

Vascular Endothelial Growth Factor (VEGF)

VEGF family currently includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PLGF). VEGF family members exert their biological functions by binding to three different transmembrane tyrosine kinase receptors, designated VEGFR-1, VEGFR-2, and VEGFR-3. anti-VEGF antibody bevacizumab (Avastin, Macugen, Lucentis) is increasingly used to treat advanced malignancy and Macular Degeneration. Because of essential role of VEGF in wound healing, anti-VEGF therapies have been suggested to potentially lead to wound-healing complications (WHCs). These risk seems higher with neoadjuvant than adjuvant bevacizumab use and may be decreased by extending the bevacizumab-surgery interval. Topical VEGF therapy accelerates wound healing in experimental diabetic wounds

Fibronectin

Very big big glycoprotein. Dimer. 2 large subunits. Binds to integrins The two polypeptide chains are similar but generally not identical (being made from the same gene but from differently spliced mRNAs). They are joined by two disulfide bonds near the C-termini. RGD and synergy sequence are cell binding sites Fibronectin is secreted primarily by fibroblasts as a soluble protein dimer and is then assembled into an insoluble matrix in a complex cell-mediated process. Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation, Fibronectin is important for wound healing. Along with fibrin, plasma fibronectin is deposited at the site of injury, forming a blood clot that stops bleeding and protects the underlying tissue Altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer and fibrosis.

Wee1 and Cdc 25 with M-Cdk

Wee1 and Cdc25 represent checkpoint enzymes that limit M-Cdk action until DNA replication is complete. A replication fork protein blocks cdc25 activity Cdc25 must work to remove phosphate and allow for cycle to continue

Prader Willi and Angelman Syndrome

both have 15q imprinting region PW: 70% -- Deletion of region of paternal origin 25% -- Two copies of maternal region (Maternal UPD) 5% -- Methylation of PWS-ICR on paternal chromosome Consequence: Loss of expression of paternal-origin alleles in region Angelman: 70% -- Deletion of region of maternal origin 5% -- Two copies of paternal region (Paternal UPD) 5% -- Lack of methylation of PWS-ICR on maternal chromosome 20% -- Loss of function point mutation in UBE3A gene (ubiquitin protein ligase 3) Consequence: Loss of expression of UBE3A gene of maternal origin

Sequence

eg. Potter Sequence Renal agenesis (malformation)--> Lack of amniotic fluid (cant pee)--> Mechanical pressure on face, ears, hips, feet (deformation) --> Lack of fluid for fetus to breathe in --> pulmonary hypoplasia (lethal)

Syndromology

multiple anomalies with a single basic cause which occur independently rather than sequentially Named after an affected individual = "Name's syndrome" Named after the unaffected reporter = "Name syndrome" Eponyms usually dropped as underlying cause discovered Dysmorphology provided systematic description of physical phenotype and aided in understanding of human development

P27 with G1-S Cdk

p27 and p21 are checkpoint proteins that limit G1/S-Cdk and S-Cdk action if there is DNA damage & p53 induction