Cell cycle and its regulation

-proto-oncogenes and

tumoursupressor genes

Relation tumor-supressor genes and proto-oncogenes

- they collaborate in close mutual interaction

- their relationship is relatively antagonistical

- in reality in anyone „step“ of CC by them coded proteins(„products“) reciprocally interact:

Proto-oncogeneProto-oncogenecoded protein

TSG coded protein

prolipheration (cells division a differentiation)

Regulation of cell cycle (CC) = balance betweencyclins and (cdk´s).

products products of tumorof tumor--suppresor genessuppresor genes

products of protoproducts of proto--oncogenesoncogenes

Proto-oncogenes

- inhibit apoptosis

- are genes and their products (proteins), that:

- stimulate progress in CC

- inhibit differentiation

- after their “activation “ they “switch“ to oncogenes

Protoooncogenes(in regard to position and function of their proteins

in cell):

4

1

2

3

1. Mitogens (growth factors)(e.g. PDGF, EGF, IGF, IL-2 )

2. Membrane proteins(receptorsand G-proteins)(e.g. ras, c-src, c-abl)

3. Plasmatic transmitters= signal transducing proteins(e.g. tyr., ser. & threonine kinases)

4. Nuclear – mainly transcription factors(e.g. C-fos, jun, ets, myc, erbA, erh-B)

Signal transduction and CC

A) What is signal transduction?

B) What are growth factors?

C) How do they contribute to normal signal transduction (ST)?

Altered Metabolism

MetabolicEnzyme

Gene Regulator Cytoskeletal Protein

Change ofGene

Expression

Altered Cell Shape or Motility

Adapted from Molecular Biology of the Cell,(2002), 4th edition, Alberts et al.

Signaling Pathway

SignalSignal

Receptor (sensor)

Transduction cascadeTransduction cascade

TargetsTargets

ResponseResponse

Simple signal transduction

Growth Factors

- proliferation- differentiation- growth- survival - angiogenesis(growth of tumor blood vesels)

– ligands which bind enzyme linked receptorsligands which bind enzyme linked receptors

– signal diverse cellular responses including:

- can impact multiple cell types or is cell-specific

Factor Principal Source

Primary Activity Comments

PDGF platelets, endothelial cells, placenta

promotes proliferation of connective tissue, glial and smooth muscle cells

two different protein chains form 3 distinct dimer forms; AA, AB and BB

EGF submaxillary gland, Brunners gland

promotes proliferation of mesenchymal, glial and epithelial cells

TGF-α common in transformed cells

may be important for normal wound healing

related to EGF

FGF wide range of cells; protein is associated with the ECM

promotes proliferation of many cells; inhibits some stem cells; induces mesoderm to form in early embryos

at least 19 family members, 4 distinct receptors

NGF promotes neurite outgrowth and neural cell survival

several related proteins first identified as proto-oncogenes; trkA (trackA), trkB, trkC

Erythropoietin kidney promotes proliferation and differentiation of erythrocytes

TGF-β activated TH1cells (T-helper) and natural killer (NK) cells

anti-inflammatory (suppresses cytokine production and class II MHC expression), promotes wound healing, inhibits macrophage and lymphocyte proliferation

at least 100 different family members

IGF-I primarily liver promotes proliferation of many cell types

related to IGF-II and proinsulin, also called Somatomedin C

IGF-II variety of cells promotes proliferation of many cell types primarily of fetal origin

related to IGF-I and proinsulin

Growth Factorsexamples

Growth Factor ReceptorsGrowth Factor Receptors

Dimerisation = activation of receptor = transfer of signal

Most Most growth factors growth factors bind bind to tto tyrosine yrosine kkininaasessesrr eceptor eceptor (RTKs(RTKs))

- extracellular domains are extremly variable = similar or unique= some GF are „universal“ and other GF are specific

-- intracellular domains are almost uniform onesintracellular domains are almost uniform ones

Characteristics of the Common Classes ofRTKs

Class Examples Structural Features of Class

IEGF receptor,NEU/HER2, HER3 cysteine-rich sequences

II insulin receptor,IGF-1 receptor

cysteine-rich sequences; characterized bydisulfide-linked heterotetramers

III PDGF receptors,c-Kit

contain 5 immunoglobulin-like domains;contain the kinase insert

IV FGF receptorscontain 3 immunoglobulin-like domains aswell as the kinase insert; acidic domain

V

vascularendothelial cellgrowth factor(VEGF) receptor

contain 7 immunoglobulin-like domains aswell as the kinase insert domain

VI

hepatocyte growthfactor (HGF) andscatter factor (SC)receptors

heterodimeric like the class II receptorsexcept that one of the two protein subunitsis completely extracellular. The HGFreceptor is a proto-oncogene that wasoriginally identified as the Met oncogene

VII

neurotrophinreceptor family(trkA, trkB, trkC)and NGF receptor

contain no or few cys teine-rich domains;NGFR has leucine rich domain

Characteristics of common clases of RTKsCharacteristics of common clases of RTKs

Growth Factor Receptor Activation

RTKReceptorSubstrate/TK

Signal transductionthrough tyrosin kinase receptor

Overview of Overview of mitogene activated protein kinanses (mitogene activated protein kinanses (MAPKMAPK s)s) ssignaling ignaling ppathwaysathways

ProtoProto--oncogenes that oncogenes that eencode ncode ssignalling ignalling pproteinsroteins

Serine/Threonine Kinasesc-raf familyakt

Non-receptor Tyrosine Kinasessrcabl

Receptor associated binding proteinsc-ras family

• Active Ras interactswith the first kinase in the MAPK cascade, Raf.

• It localizes Raf to the membrane, where it is activated by an unknown mechanism

• This starts the cascade

Activation of the mitogene activated protein kinases (MAPK) cascade

• Each kinase in the cascade is activated by phosphorylation in a regulatory site, called the t-loop

• When T-loop is phosphorylated, a conformation change occursand the catalytic cleft is “opened” and active

• Each kinase is bound by modifying enzymes(incoming signals).

• The three kinases may be bindtogether in one complex with the MP1 scaffold protein

Activation of the mitosis activating protein kinases (MAPK) cascade

The Rasand Shc-Ras activated MAPK is the target of allErbB ligands.

Signal Processing

MAPK targets

• The MAPK phosphorylates and activatesmany different targets

• For example, after phosphorylation it may translocate to the nucleus andactivate transcription factors

• It also phosphorylates the receptor kinase and other enzymesin the pathway in an inhibitory fashion (negative feedback)

Proto-oncogenescan be convertedto oncogenesoncogenesby:

1. 1. MutationMutation

2. 2. OverexpressionOverexpressionof geneof gene::

= normal amount of abnormal protein= normal amount of abnormal proteinproduct product (sending false signals)(sending false signals)

= abnormally increased = abnormally increased amount of normal proteinamount of normal proteinproduct product (= too strong action)(= too strong action)

1.

Proto-oncogenescan be convertedto oncogenesoncogenesby:

1. 1. MutationMutation = normal amount of abnormal protein= normal amount of abnormal proteinproduct product (sending false signals), (sending false signals), e.g.:e.g.:

B) translocation = e.g of chromosomes 9 and 22 in CML(chronic myeloid leukemia) creates fusiongene bcr-abl coding permanently activeabl MAP-kinase sending false signalsto nucleus

A) point mutation = e.g. ras protooncoge codes protein sending to the cell false signalswithout activation of receptor for GF

2.

Generation of the Philadelphia chromosome Generation of the Philadelphia chromosome observed in >95% of chronic myelogenous leukemias (CML). observed in >95% of chronic myelogenous leukemias (CML).

Proto-oncogenescan be convertedto oncogenesoncogenesby:

2. 2. OverexpressionOverexpressionof geneof gene= abnormally increased amount of normal protein (= too strong action)

A) regulatory = too high expressionof normal amount of alelles

3.

B) amplification = increraseof alelles amount

a) cellular = e.g. translocation of PrO under strong enhancer action like c-myc in Burkitt lymphoma

b) retroviral = insertion of RV provirus before (till 3kb) of PrO promoter (its LTR sequences)increases PrO expression = s.c. non-acute transforming RV

a) cellular = e.g. unequal crossing-over, „healing“ and replicationof broken chromosome = see textbook

b) retroviral = RV gained (by translocation with cellular mRNA in lytic part of

RV cycle)viral copy of PrO (s.c. v-onc) and it is extremelly expressed = acute deregulation of CC = s.c. acute transforming RV

Retroviruses in etiology of cell malignant transformation

Retroviruses

- the genome of RV is simillar to mRNA = high risk of recombinationwith mRNA arrising from c-onc!

GpppG

cap

R -U5

att

PB

5´ 3´

PP

líder

att

promótor

poly A signál

AnU3-R

regulačné sekvencie kódujúce

sekvencie

regulačné sekvencie

gag pro pol env

genóm retrovírusunapr. HIV) má ako genóm +ssRNA Genome of RV

regulatorysequences

regulatorysequences coding

sequences

Some retroviruses could contain additional genetic information = v-onc

Some retroviruses could contain additional genetic information = v-onc

R U5 GAG POL ENV U3 R

“typical“ retrovirus genome

Rous Sarcoma virus genome

R U5 GAG POL ENV U3 RSRCSRC

= no loss of own genes !!!!!= no loss of own genes !!!!!

= are genes coding proteins:

- stimulating apoptosis

- inhibiting progress of CC

- supporting differentiation

Tumor suppressor Tumor suppressor ggenesenes

TumorTumor --supressor supressor genes genes can take part inCC deregulationCC deregulationby:

1. 1. MutationMutation

2. 2. Blocking Blocking of by them coded normal proteins activityof by them coded normal proteins activity::

= is lost (deleted or mutated) of last working= is lost (deleted or mutated) of last workingalelle of TSG = mostly after alelle of TSG = mostly after LOH LOH (loss of heterozygosity)(loss of heterozygosity)

= e.g. by oncoproteins of DNA viruses= e.g. by oncoproteins of DNA viruses

1.

TumorTumor --supressor supressor genes genes can take part inCC deregulationCC deregulationby:

1. 1. MutationMutation = is lost (deleted or mutated) last working= is lost (deleted or mutated) last workingalelle of TSG = mostly after alelle of TSG = mostly after LOHLOH

2.

B) somaticmutation = happens in single body (= somatic) cell and it is present only in sub-population of daughter cells.

Probability of second mutation = LOH is very low.Often it is called as „sporadic“origin of malignities.

A) gameticmutation = one gamet brings mutation and second one will bring normal alelle = eachbody cell is heterozygous! If is last workging alelle mutated =LOH.

Probability of second mutation = LOH is high.No more working TSG protein is present in the cell.

Often it is called as familial origin = „inherited“ (Mendelian) malignities.

Protein rb1

Р

pRB

E2F1

PРP

PРP

E2F1

pRB

Cyclin D/CDK4 Cyclin Е/СDK2

PР

P

PР

P

PР

P

PР

P

PР

P

DNA synthesis not allowed

DNA synthesis allowed

Cyclin complexes are not active

pRB less phosphorylated

E2F1 bound to pocket

Cyclin complexes are active

pRB more phosphorylated

E2F1 is free to promote cell cycle

Four Ways to Inactivate RBFour Ways to Inactivate RB

1. Mutation of RB gene. 1. Mutation of RB gene.

2. Binding by DNA viral oncoproteins.2. Binding by DNA viral oncoproteins.

3. Cdk-mediated phosphorylation.3. Cdk-mediated phosphorylation.

4. Caspase-cleavage and degradation.4. Caspase-cleavage and degradation.

Cell cycle progressionCell cycle progression……..

Rb-/- neurons and muscles undergo apoptosis.

E1A induces apoptosis.

Activated by apoptosis inducers, TNF.

TumorTumor --supressor supressor genes genes can take part inCC deregulationCC deregulationby:

2. 2. Blocking Blocking of by them coded normal proteins activityof by them coded normal proteins activity::= e.g. by oncoproteins of DNA viruses= e.g. by oncoproteins of DNA viruses

3.

Principle is in fact, that DNA polymerases are present in eukaryotes cells only after succesfull passing through G1 check point!

BUTBUT DNA viruses need those enzymes to reproduce their DNA =by their oncoproteins = they can is in fact destroy G1 check point (by „extracting“ represor protein rb1) ...

Viral oncoproteins Viral oncoproteins can interact with RB1 and occupate its pocket

Р

pRB

PРP

PРP

E1A, SV40-TAg, HPV-E7

E2F1

Adenoviral E1AE1A,Large TLarge Tantigene of SV40,E7E7protein of HPV type 16 or18

Viral oncoproteins bind to pocket domain of rb1

Set E2F factors free !!!!!

Cell is going to proliferateCell is going to proliferatedespite low phosphorylation of rb1 !!!!despite low phosphorylation of rb1 !!!!

“ Retinoblastoma is a cancer caused by two mutational events. In thedominantly inherited form, one mutation is inherited via the germ cell and the second occurs somatically. In the nonhereditary form,both mutations occur somatically.”“ Using Poisson statistics, one can calculate that this number (three) canexplain the occassional gene carrier who gets no tumor, those who developbilateral tumors, as well as explaining instances of multiple tumors in one eye”

Proc. Natl. Acad. Sci. USA 68, 820-823 (1971)

Alfred G. Knudson Jr . - statistical study of retinoblastomaretinoblastoma:

LOH = loss of heterozygosity

Protein p53

Ternary complex of p53Ternary complex of p53

cytoplasmcytoplasm

nucleusnucleusMdm2

Cell cycleCell cyclearrestarrest

DNA damage repairDNA damage repair

ApoptosisApoptosisBax Fasp21Waf1/Cip1 Gadd45

p53p53p

pp pp

p pp

p53Mdm2p

ppp

ser15ser20

ser37ser392

Wild type p53Wild type p53

Protein p53

In Li-Fraumeni syndrome has individualin each body cell mutated one allele of TP53gene

(as a result of gametic mutation).

- mutations are accumulated in cells(= appear benign tumours) and it is only a “question of time“ when, after LOH in TP53, become certain cell malignant

= p53 produced by his (her) (heterozygous) cells is not able to carry out its basic function= start apoptosis (if inreparable amount of mutations).

= each person with sy. Li-Frameni need to have in mind, thatdo have 50 % probability to transfer mutation to child (without speaking about chance to care about child...).

WT1WT1

- protein WT1 is regulator of transcription of genes taking part indevelopment of kidney and gonads

Gene WT1 is coding DNA binding regulatory protein (DNAbp)sc. “zinc finger“ working as activator or repressor of transcription

- if mutated = takes part in origin of Wilm ´s tumour

NFNF--11Neurofibromatosis 1(NF-1) gene (17q 11.2) has more than 300 kb of genomic DNA, 50 exons

- function of NF1 is inactivation of proto-oncogene ras

APC APC = Adenomatous polyposis coli= Adenomatous polyposis coli

APC geneis located at 5q21 and its mutations appear in FAP(familial adenomatous polyposis coli), in Gardner syndrome e.t.c.

- tumor suppressor protein APC works as “gatekeepergatekeeper“of intestine epithelium proliferation

- in cytoplasm APC (after signal from morphogene Wnt) release beta-cateninand enter nucleus to start expressionof proper genes

- normal product of APC gene takes also part in origin ofintercellular connections

APCAPC

- in locus 5q21 is gene DP2.5 „working as APC“

- nowadays are known more than 120 mutations of this gene

DCC DCC = deleted in colorectal carcinoma= deleted in colorectal carcinoma

- gene is located at 18q21.3, close to telomera

- DCC protein has structures common for proteins taking partin adhesive connections of cells and with other proteins collaborates at interactions cell-cell or cell-extracellular matrix

- loss of allele(LOH) of DCC (but also terminal deletion of 18q) has negative consequences for patient with colorectal cancer = increase probability for beginning of metastasis processbeginning of metastasis process

- gene DCC also code netrin receptor, required for „guiding“of axons (in vitro, too)

- protein coded by DCC gene has morphogeneticrole in formation of contacts (axon-cell) between neurocytes = in “targeting“ of these cells and their processes

Carcinoma development and invasion