Download Objectives 25

INTRODUCTION
- proliferation of cells in multicellular organisms is regulated with a fine balance of stimulatory
and inhibitory signals; defects in this mechanism that mediate this balance can cause cancer,
which uncontrolled growth induced be either loss of function in inhibitory signals (tumor
suppressors) or gain of function in stimulatory signals (oncogenes)
REGULATION OF THE EUKARYOTIC LIFE CYCLE
- mitosis (M phase) and DNA synthesis (S phase) are conspicuous  distinct and identifiable
events occur in nucleus during each of these phases
- S follows M phase by G1 phase and M follows S by G2
- prior to S phase  cells are diploid; after S phase  cells are tetraploid; all non-cycling cells
in humans are diploid; physiological growth arrest occurs during G1 phase
- entry into each phase regulated by cell cycle checkpoints; cell will arrest if checkpoint
requirements not met; most stringent checkpoints are prior to S and M phases
Cell cycle checkpoints
- G1 checkpoint: ensures cell is prepared to complete a round of DNA synthesis.
- Most physiological growth arrest occurs here, as most cells in the body are 2n.
- G2 checkpoint: ensures cell is prepared to undergo mitosis  requires microtubules
- Checkpoints are associated with cyclins.
Cyclins and Cyclin-Dependent Kinases (CDKs)
- progression through phases of cell cycle associated with increased expression of cyclins 
synthesized de novo in each cell cycle phase and degraded by proteosome following phase
completion (ubiquitin mediated degradation)
- specific cyclins associated with specific phases; cyclin D1 associated with G1 phase, cyclin A
with S phase, cyclin B with G2/M phase, cyclin E with G1/S boundary
- cyclins exists a part of multi-protein complexes that contain cyclin-dependent kinases
(CDKs); specific CDKs associate with specific cyclins, such as CDK-2 with cyclin A/E, CDK-4
with cyclin D1; CDKs phosphorylate specific protein substrates on serine or threonine
residues, which regulates substrate activities
- resting cells (non-cycling) are diploid; most important point in cell cycle regulation occurs
prior to initiation of S phase
- if a cell enters S phase  it is committed to divide; this point is called START and is defined
as the committed point after which DNA synthesis will ensure and cell will divide
- GFs removed from cells  cell cycle arrests at a restriction (R point), prior to START
- to traverse R-point and proceed to S phase  G1 complex must be formed which contains
CDK-4, PCNA, and Cyclin D; complex is active when CDK-4 phosphorylates protein pRb
(tightly bound to a TF called E2F); once phosphorylated, prb-E2F complex dissociates and
E2F activates set of genes needed for DNA replication (DNA polymerase); genes are START
genes
- pRb is an inhibitor of cell cycle  tumor suppressor gene; pRb defect  incidence of
retinoblastoma at early age; tumor suppressor mutations are loss of function mutations
- other tumor suppressor (inhibitor of cell cycle)  p21WAF associates/inhibits kinase activity of
complex
Regulation of the cyclin D/CDK4 checkpoint
- can be activated by growth promoting signals such as epidermal growth factor (EGF) or
inhibited by negative growth modulators, such as TGF-beta, p27, and p15 by DNA damage via
p53 induction of p21WAF or by E2F itself (which negatively feedbacks via activation of p16_
- p53 (master switch tumor suppressor gene); it is normally unstable but stabilized by ATM
(protein responsible for ataxia telangiectasia)
- ATM is a kinase activated by DNA damage (caused by ionizing radiation)
- p53 induces p21WAF (cell cycle arrest); it also stimulates GADD-45 (growth arrest and DNA
damage inducible protein-45)  initiates DNA repair enzymes; p53 sense DNA damage 
arrests cell cycle  induces DNA repair
- lose p53 function  accumulation of DNA mutations (cancers); p53 mutated in 60% of all
human cancers; any mutated p53 inhibits holoenzyme in dominant negative fashion
- p53 continuously synthesized/degraded; degradation accomplished by a ubiquitin ligase,
mdm-2  adds multiple copes of ubiquitin onto p53  polyubiqiunated p53 targeted for
degradation by proteosome
GROWTH FACTORS AND SIGNAL TRANSDUCTION
- cells require growth factor hormones to proliferate; absence of GF  normal cells arrest at
R-point of cell cycle; add GF (EGF)  DNA replication
Growth factor-receptor interaction
- peptide GF bind to receptors which contain tyrosine kinase domains; these kinase domains
autophosphorylate tyrosine residues on the cytoplasmic side of receptor  induce specific
binding of proteins which contain a src-homology region (SH2); SH2 domains have high
affinity for phosphotyrosines  phosphorylated cytoplasmic tail of GF receptor serves as
anchor to which SH2-containing proteins bind/interact
The ras and raf signaling pathway
- Shc protein contains both SH2 and phosphotyrosine domains and docks GRB-2 to
phosphotyrosine-containing receptor
- GRB-2 (growth factor receptor binding protein) is a SH2-containing protein which activates
SOS, a guanine nucleotide exchange protein which activates ras; ras is a small G-protein that
is activated by binding of GTP; ras  activates raf (cytoplasmic ser/thr kinase)  activates
the MAP kinase cascade
- intrinsic GTPase activity inactivates ras by hydrolyzing GTP  GDP; inhibition of activity
leads to carcinogenesis
Map kinase cascade
- MAP (mitogen activated protein) kinases are a series of serine-threonine kinases that
phosphorylate one another in a cascade  amplifies signals and provides a point of integration
because these kinases can be activated by signals other than the EGF signaling event; at end 
MAP kinase phosphorylates nuclear TFs (Elk-1, Jun)  induce proliferation-associated gene
transcription (START)
GROWTH FACTOR FAMILIES
- distinguished by structure, receptor structure, and cells specificity
Class IA: EGF – Epidermal Growth Factors*
- major forms are 6 kD
- stimulates proliferation in epithelial, mesenchymal, and glial cells
Class IB: GF – fibroblast growth factors (FGF)
- acidic (16 kD) and basic (17 kD); FGF proteins are found associated with ECM and stimulate
proliferation of endothelial, epithelia, mesenchymal and neuronal cells
Class II: IGF- insulin-like growth factors*
- 7 kD; include IGF-1 and IGF-2; related to proinsulin structurally will activate insulin
receptor at high concentrations
- IGf-1 produced in liver; IGF-2 produced by tumor cells; both present in plasma in
conjunction with specific binding proteins
- IGF stimulate glucose metabolism; they also stimulate proliferation and inhibit apoptosis
Class IIIA: PDGF – Platelet-derived growth factors*
- mitogenic GFs occurs as dimers of A (17 kD) and B (16 kD) chains
- heterodimers (AB) and homodimers (AA, BB) exist
- PDGF is derived from platelets and endothelium in adult tissues and from placenta
- PDGF stimulates proliferation in mesenchymal, glial, and smooth muscle cells
Class IIIB: Cytokinase/GH
- interleukins (IL1-IL6), erythropoietin, and growth hormone are not GFs
- they are classed as cytokines and regulate differentiation
- exists as homo/hetero dimers; activate receptor that does not have intrinsic tyrosine kinase
activity
GROWTH FACTOR RECEPTORS
- growth factor receptors contain tyrosine kinase activities and are known as receptor tyrosine
kinases (RTK); distinct from type IIIB Janus-associated tyrosine kinase receptors (JAK) 
recruit tyrosine kinases upon activation
- RTKs are specific for polypeptide growth factor hormones  work in autocrine or
paracrine fashion  stimulate local proliferation in target tissues
- three families of RTKs: I, II, and III; all activated by dimerization; Types I (EGFR) and III
(PDGFR) are monomers in native (unbound) forms; types II (IGFR) is a dimer in native
form
Classes of RTKs
- 3 classes of RTKs  EGF-R, IGF-R, and PDGF-R
- types I/II have one TK domain per chain; type IIIA has two TK domains per chain
- type IIIA  immunoglobulin-like repeats
- receptor class II (insulin receptors) are dimers in native state; the ligand binding domain of
the class II receptors is not imbedded in membrane, but is attached to the transmembrane
subunit through a disulfide linkage
- other two classes exist as monomers in their native form and are activated by ligand-induced
dimerizaiton
Dimerization
- essential for receptor activation  results in cross-phosphorylation by adjacent chains
- overexpressed mutant receptors inhibit native receptors in dominant negative fashion
- class I/III  receptors are phosphorylated and the phosphotyrosines provide binding sites
for SH2 domain-containing proteins
- class II RTKs  IRS-1 (insulin receptor substrate) is poly-phosphorylated to provide SH2
binding sites
ONCOGENES
- tumorigenic transformation caused by either viral or chemical agents
- theory: cells contain a set of genes called proto-oncogenes  involved in regulation of normal
proliferation; chemical carcinogens disrupts the regulation of these genes/gene products 
cancer causing oncogene results
- other proposal: viral gene products are either homologous to oncogenes or disrupt normal
proto-oncogene function (viral oncogene  src gene for sarcoma virus, but no cellular
homolog to this viral gene product found)
- cellular homolog to avian MC29 myelocytomatosis virus myc oncogene found
- proto-oncogenes usually involved in proliferation regulation; most identified by homology
to viral oncogene products
- 6 classes of oncogenes (based on activity); proto-oncogenes themselves are not transforming
and essential for normal growth
Classes of oncogenes
- class 1/2  growth factors and their receptors; function in growth control
Class 1 oncogenes – growth factors
- sis, TGF-alpha (EGF); TGF-alpha is secreted by many cell types (autocrine GF); sis
secreted by sarcomas  growth of sis-secreting sarcomas in vivo is suppressed by
neutralizing antibodies against the sis protein showing that secretion/activity of this oncogene is
necessary necessary/sufficient for unregulated growth
Class 2 oncogenes – growth factor receptors
- erbyB/her2-neu oncogene is an EGF receptor; when activated through overexpression  this
oncogene is expressed in cancers (breast)  constitutively active and do not require GFs
Class 3 oncogenes – non-receptor (cytoplasmic) tyrosine kinases
- bcr-abl (created by chromosomal translocation)  moves abl gene adjacent to an
immunoglobulin promoter
- immunoglobulin producing B-cells overexpress abl; immunoglobulin producing B-cells
overexpress abl and lose their growth control  B-cell leukemia (chronic myelogenous
leukemia CML) results; abl inhibited by Gleevec
Class 4 oncogenes – small G-proteins
- small G-proteins prevalent in oncogenesis; ras is common  mutated in 60% of cancers; it
shares with p53 the distinction of being the most commonly observed mutation in cancer; two
major classes of oncogenic ras proteins identified by homology to Harvey (H-ras) and Kirsten
(K-ras) sarcoma viruses
- mutation occurs at a specific sites which inactivates GTP hydrolyzing activity  protein then
becomes constitutively active; ras activates downstream MAP kinase cascade through raf
Class 5 oncogenes – cytoplasmic serine/threonine kinases
- c-raf  initiates the MAP kinase cascade; mitogenic stimulation by GFs  increase in
phosphorylation of c-raf  increase in c-raf activity
- oncogenic forms of raf have lost N-terminal sequences that are regulatory  leading to
permanent activation and altered localization
Class 6 oncogenes – nuclear transcription factors
- nuclear proto-oncogenes are TFs which bind and activate specific promoter elements in
DNA to induce transcription of specific families of gene products
- in oncogenic forms  these factors lose regulation or activate transcription of abnormal
genes
- myc is a nuclear proto-oncogene; V-myc is the transforming agents in MC29 avian
myelocytomatosis virus
- mitogenic agents induce expression of c-myc in most cell lines
- myc has homology to other TFs with helix-loop-helix and leucine zipper motifs
- myc dimerizes with protein max prior to DNA binding and myc is involved in regulating
apoptosis
TUMOR SUPPRESSOR GENES
- mammalian cell growth is balance between growth promoting signals and growth inhibiting
signals
- growth promoting signals  GF signal transduction; mutation result in gain-of-function
(constitutively active) mutation (in proto-oncogene) and gives rise to oncogenes  cancer
- growth inhibitory signals observed at cell cycle checkpoints ; loss-of-function mutations in
these signals result in reduced tumor suppressor activity and cancer
- loss-of-function more common than gain-of-function
-patient has defect in one tumor suppressor allele  loss of 2nd allele occurs with high frequency
 complete loss of activity loss of heterozygosity (LOH)  prominent mechanism in tumor
suppressor mediated tumorigenesis
- greater loss-of-function prevalence  number/type of tumor suppressor mutations is broader
than those for oncogenes
- any gene that promotes well-regulated growth is a tumor suppressor
- major tumor suppressor is p53  designed to protect cells against DNA double strand breaks
(UV or oxidation caused); it is a TF that arrests cell cycle and induces DNA repair
- if DNA damage not repaired in a given amount of time  p53 can induce apoptosis and
caspase activity