How the tumor is initiated ?
• 1. Chemical carcinogenesis
• 2. Hormonal carcinogenesis
• 3. Viral carcinogenesis
• 4. Bacterial/parasitic carcinogenesis
• 5. Chronic inflammation carcinogenesis
• 6. Spontaneous carcinogenesis
as a sum of all things above
TEST
Carcinogen
Any agent that produces cancer, e.g.
tobacco smoke,
certain industrial chemicals,
ionizing radiation
(such as X-rays and ultraviolet rays).
1. Chemical carcinogenesis
Carcinogens
1. Genotoxic
(direct DNA damaging carcinogens)
produce DNA adducts;
one application is enough for tumor initiation
2. Non-Genotoxic
(damage DNA as result of secondary interactions,
e.g. increase of oxidative stress, inflammation)
Genotoxic Carcinogens – mutators
Anti-tumoral
chemotherapeutic drugs
(cyclophosphamide,
busulfan, chlorambucil),
beta-propiolactone
Acetylating and alkylating
agents
Direct carcinogens
(no modification needed)
Aromatic Amines and Azo Dyes
Pro-carcinogens
that have to be modified
by intracellular enzymes
benzanthracene
(first pure carcinogen)
3,4-benzpyrene
(isolated from coal tar)
Aflatoxin B1
7,12-dimethylbenzanthracene
(most potent carcinogen)
Benzo[a]pyrene
polycyclic aromatic
hydrocarbons (PAHs)
is initially oxidized, primarily by the
microsomal NADPH- dependent
cytochrome P-450,
to several arene oxides.
Benzo[a]pyrene itself can bind AR
(Aryl hydrocarbon receptor) and
activate gene expression:
cytochromes,
MAP kinases,
IGF-1 (insulin-like growth factor)
Benzo[a]pyrene derivates
can bind and damage DNA.
Benzo[a]pyrene
cytochrome P-450
arene oxide
certain strains of the fungi
Aspergillus flavus
and A. parasiticus
oltipraz, an inducer of
aflatoxin metabolizing enzymes,
significantly increased biomarkers
of aflatoxin detoxification
Prevention trial in China
Aflatoxin B1
Non-Genotoxic Carcinogenes:
Tumor Promotors
DMBA Olive oil - 6 months
DMBA Croton oil - 6 months
NO tumors
Multiple
tumors
DMBACroton oil - 6 months
NO tumors
Promotor =
Promotor =
Two-phase carcinogenesis of mouse skin
TUMOR PROMOTERS
1. Promoting agents are not carcinogenic per se
2. Can promote cancer after very small doses
of initiating (true carcinogenic) agents
3. Promoting agents can wake tumors up
long time after administration of initiating agent
Phorbol esters
Aplysiatoxin (algal toxin)
Teleocidin (fungal toxin)
Hormones (estrogen) Growth factors
Those substances promote growth
of existing tumor clones evolved after mutation events
Phorbol ester (from croton oil)
12-O-tetradecanoylphorbol-13-acetate (TPA)
TPA is cell-toxic, pro-inflammatory agent
that increase vascular permeability
TPA activates protein kinase C
TPA induces INFLAMMATION,
and pushes epithelial cells to propagate
Pre-existing Tumor cell got her chance to grow
Professions and industries
associated with high risk of cancer
Aluminium industry polycyclic aromatic
hydrocarbons (PAHs)
Lung and bladder
cancer
Coal industry polycyclic aromatic
hydrocarbons (PAHs)
Lung, bladder, skin,
scrotum cancer
Shoemaking Benzene Lymphomas, leukemias
Furniture making Wood dust Nasopharyngeal cancer
Fuchsin dye production Fuchsin, ortho-toluidine Bladder cencer
Rubber industry Aromatic amines,
solvents
Lung, colon, stomach,
bladder, prostatic
cancer, leukemia
How to block carcinogen-dependent
tumorigenesis
• 1. block carcinogen uptake into body/cells
• 2. Inhibition of carcinogen
formation/activation by blocking Cyp450
• 3. Stimulate carcinogen deactivation by
conjugation (NAT2, GST, UGT…)
• 4. Inhibition of DNA adduct formation
(antioxidants)
• 5. Stimulation of DNA repair
2. Hormonal carcinogenesis
• 1. Estrogens
- stimulate proliferation of epithelial cells;
- estrogen metabolites are genotoxic;
• 2. Xeno estrogens
- DDE and other insecticides structurally similar to
diethylstibestron;
- Genistein from Soybean and other phytoestrogenes;
- Digitalis, Sulfonamide antimicrobials,
- Oral contraception, Hormone Replacement Therapy
(estrogen should be counterbalanced by progesteron)
Cancer site Hormones Potentially important
genes
Breast Estrogen,
progesterone
CYP17, CYP19,
HSD17B1, ER, PR
Prostate Dihydrotestosterone CYP17, HSD17B3,
SRD5A2, AR
Ovary FSH,
progesterone
FSH, FSHR, PR
Endometrium Estrogen CYP17, HSD17B1,
HSD17B2, ER
Testis In utero estrogen CYP17, HSD17B1
Thyroid TSH, estrogen TSH, CYP17,
HSD17B1
3. Viral carcinogenesis
• 1. DNA containing oncoviruses
-- Polyomaviridae
SV40 (monkey, hamster)
Polyoma (mouse)
JC and BK viruses (hamster)
-- Papillomaviridae
Human papillomavirus (HPV 16, 18)
cervical carcinoma
-- Adenoviridae
types 12, 18, 31, 3, 7,14 (hamster)
-- Poxviridae
myxomavirus (rabbit)
Jabavirus, tanapoxvirus (benign skin histiocytoma),
contagiuos mollusc virus (benign pearl-like skin
tumors, 5% of Pacific population)
3. Viral carcinogenesis (cont).
• 1. DNA containing oncoviruses
-- Herpesviridae
Epstein-Barr virus (EBV)
Hodgkin disease, nasopharyngeal carcinoma
Kaposi’ sarcoma virus (KHSV)
Marek disease virus (chicken)
American rabbit virus
-- Hepadnaviridae
Hepatitis B virus (liver tumors)
Woodchuck hepatitis virus
Duck hepatitis virus
3. Viral carcinogenesis
• 1. RNA containing oncoviruses
-- Alpharetrovirus
Rous sarcoma virus (RSV),
Chicken lympholeukosis
-- Betaretrovirus
Mouse mammary tumor virus (MMTV)
-- Gammaretrovirus
Moloney sarcoma virus (mouse)
Feline sarcoma (FSV)
--Deltaretrovirus
Bovine leukosis;
Human adult T-cell leukemia virus (HTLV)
Retroviruses
(best understood oncogenic viruses)
Peyton Rous
Born Baltimore (Maryland)
1866-1970
1966 –Nobel Prize
“for his discovery
of tumour-inducing viruses"
1909 Rockefeller Institute
Chicken sarcoma could be "transferred"
into a healthy chicken by grafting tumor cells.
Cell-free filtrates from the tumor
also led to sarcomas in healthy chickens.
By 1914, Rous's laboratory had discovered
three distinct types of avian sarcomas.
Virus = “filterable agent”
"minimal"
retrovirus
contains
Env
Gag
Pol
POL = reverse transcriptase;
RNADNA http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/
Scheme of retrovirus:
Budding a retrovirus from the cell
http://genetherapy.genetics.uiowa.edu/
Lipid envelope
is hijacked form the cell
RETROVIRUS
LIFECYCLE
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/
RNA-pol (RT) attached to
viral genomic RNA
DNA copies
Random insertions
to host genome
Producing of fresh RNAs
Translation Packaging
http://www.blc.arizona.edu/marty/411/Modules/Lectures/Figures/Trans_Retros.GIF
Most natural oncogenic viruses are defective:
not able to propagate without helper virus
that provides necessary gene products
(non defective)
(defective, lack Pol and Env genes, added Abl oncogene)
(defective, lack Pol and Env genes, added Ras oncogene)
(mouse)
Oncogenes (Viral Onc- genes)
• Oncogenes were discovered in “classic”
oncogenic viruses (retroviruses)
• (Sarcoma Rous virus) Src
• (Abelson murine leukemia  Abl
Viral oncogenes have normal cellular homologues,
that also could be tumorigenic if mutated
v-SRC, v-ABL, v-HA-RAS are derived
from host c-ONC sequences
(probably picked up as processed transcripts)
Human Adult T-cell leukemia viruses
HTLV-1 and HTLV-2
• HTLV-1 (human T-cell lymphotropic virus)
-- is sexually transmitted;
-- endemic to Japan, Caribbean
-- causes Adult T-cell leukemia
(Sezary T-cell leukemia);
HTLV-2
-- T variant of hairy cell leukemia;
-- Native Amerindian populations
seroprevalence is over
50%.
www.showa.gunma-u.ac.jp/. ../bp1-4/tsld014.htm
Human papillomaviruses HPV6 and HPV18
www.tulane.edu
Causative agents for female cervical carcinomas,
as well as for genital and regular warts
May integrate into the genome
or persist as episomal form
Gene: Function:
E1 Initiation of DNA replication (helicase)
E2 Transcriptional regulation/DNA replication
E3 ???
E4 Late NS protein; Disrupts cytoskeleton?
E5
Transforming protein, interacts with growth factor
receptors, e.g. PDGF
E6
Transforming protein, binds to p53 leading to
degradation
E7 Transforming protein, binds to pRB
E8 ???
L1 Major capsid protein
L2 Minor capsid protein
Human papillomaviruses HPV6 and
HPV18
Human hepatitis B virus
• Cause liver cancer (30-50 yrs
after infection);
• Average life expectancy after
diagnosis of liver cancer is 6
months;
• DNA virus that encodes 4 genes
Pol – DNA polymerase
Env -- envelope
pre-core – viral capsid
X - activation of host cell
genes and the development of
cancer.
www.immunize.org/images/
ca.d/ipcd1861/img0022.htm
Human hepatitis B virus
www.safetyline.wa.gov.au
/.../ l81_02.asp
Epstein-Barr virus
Cause infectious mononucleosis;
associated with:
-- lymphomas in immunosuppressed persons,
-- nasopharyngeal carcinomas
-- Burkitt lymphoma (endemic; 8 in every 100,000
children in parts of Africa and Papua New Guinea);
-- cofactor for Hodgkin disease (30 yrs after infection);
In Third World nations, most children are infected with
EBV;
In most industrialized nations, about 50% of the people
are infected.
EBV possess
a 172 kb genome
EBNAs
1, 2, 3A, 3B and 3C,
and EBNA-LP
= nuclear antigenes
EBNA-1 is involved in promoting
viral DNA replication
EBNA-2 is a transcription factor
with viral and host cell targets
LMP1 expression in rodent cell
lines
results in transformation
LMP2 associates with src
and several other tyrosine kinases
encoding 100 genes.
Kaposi’s sarcoma virus
www.kcom.edu/.../ lectures/lecture/aids.htm
HHV-8 virus,
common in AIDS patients and in
transplants recipients
Virus contains 81 genes;
including chemokines vMIP1 and vMIP11,
as well as a chemokine receptor,
oncogene GPCR and VEGF
Agent co-infecting
homosexual men along with HIV
KSHV incidence:
HIV+ men 25-30%
HIV+ women 3-4%
HIV+ haemophiliacs 2-3%
4. Bacterial and parasitic
carcinogenesis
• 1. Helicobacter pilori
and stomach lymphomas
• 2. Schistosomiasis
and bladder carcinoma
Helicobacter pylori is linked to MALT
lymphoma and gastric carcinoma
Both cellular and humoral immune responses are activated
but the bacteria still manage to persist lifelong
unless eradicated with antibiotics.
From Emad M El-Omar on-line lectures
H. pylori
overlying
the gastric epithelial cells.
H. pylori causes
more than 90% of duodenal ulcers
and up to 80% of gastric ulcers.
Treatment of gastric and duodenal
ulcers
1. Lowering of gastric acidity:
(life-long, relapse after cease of therapy)
– H2 blockers
– proton pump inhibitors.
2. 10 days to 2 weeks :
Amoxicillin or metronidazole, or clarithromycin,
plus either:
ranitidine bismuth citrate or bismuth subsalicylate
Eradication rates of the range from 61% to 94% depending on the regimen used.
Outcomes of H.pylori infection
Infected persons
have a 2- to 6-fold
increased risk
of developing
gastric tumor
Odds ratios for gastric carcinoma
MANFRED STOLTE, ALEXANDER MEINING “ The Oncologist”
Mechanisms of gastric carcinoma
induction by H.pylori
MANFRED STOLTE, ALEXANDER MEINING “ The Oncologist”
anticarcinogens
Schistosomiasis
• Schistosoma mansoni (intestinal)
• S haematobium (urinary)
• S. japonicum (intestinal)
• S. mekongi (intestinal)
• S. intercalatum (intestinal)
ehp.niehs.nih.gov/docs/ 2004/112-2/
Fresh water snail is an intermediate host.
On contact with humans, the parasite burrows into the skin,
matures into another larval stage (schistosomula),
then migrates to the lungs and liver
(where it matures into the adult form).
The adult worm then migrates to the intestine, liver or bladder
web de l'Organisation Mondiale de la Santé
It affects 200 million people worldwide,
mostly in sub-Saharan Africa
Schistosomiasis
is not usually found
in the United States.
In Egypt, schistosomiasis linked with cancer
is the primary cause of death among men aged 20 - 44.
http://www-micro.msb.le.ac.uk/224/Schisto.html
Cross-section of different human tissues showing Schistosoma sp. eggs.
Schistosoma sp. in bladder and liver, respectively
www.dpd.cdc.gov/
5. Inflammatory carcinogenesis
ALL Pro-inflammatory agents are tumor promoters
Anti-inflammatory agents
can reverse action of tumor promoters
anti-inflammatory steroids (dexamethasone)
COX inhibitors such as indomethacin, piroxicam and sulindac
Prostaglandins PGE2 and PGF2alpha
Phenobarbital that makes a foci in liver
Any type of Chronic tissue wounding –
tumor can arise on chronic ulcer, burn,
trauma site…
www.eatonhand.com/handbase/ 1497905.jpg
How the tumor is initiated ?
• 1. Sporadic tumors
(occasional cancers in pedigree, various types of
tumors, late onset)
Grandmother (mother line): breast cancer at 83.
Father: prostatic carcinoma at 78. No other cancers in pedigree.
• 2. Inherited cancer syndromes
(same type of cancer in many relatives, early onset)
Grandmother: renal carcinoma at 37.
Father: renal carcinoma at 29.
Son: angioblastoma at 8.
GENES INVOLVED IN TUMOR DEVELOPMENT
Tumor Suppressors
FRIENDS Oncogenes
FOES
Lots of
genes that
change their
status as
consequence of
malignization
?
Lots of
genes that
have
marginal
influence on
tumor
growth
Inactivated
Activated
Classical point of view
Oncogenes
-- Accelerators of cell division
-- Dominantly inherited
(one defective allele
can predispose the cell
to tumor formation)
-- STOP for apoptosis
Tumor suppressors
-- Inhibitors of cell division
-- Recessive
(Mutation in one allele
predispose human to
cancer, but do not cause it)
-- HELP for apoptosis
WAYS of STATUS CHANGING
Oncogenes
-- Activating point mutations
-- Translocation under strong promoter
-- Amplification
-- Overexpression
Tumor
suppressors
-- Inactivating point mutations
-- Promotor methylation
-- Gross chromosomal deletions
-- Underexpression
MULTISTEP MODEL
of the human cancer development
MALIGNANCY MOLECULAR
EVENTS
LEUKEMIA,
chronic
2-3
LEUKEMIA,
acute
3-4
CARCINOMA,
in situ
3-4
CARCINOMA,
metastatic
5-12
One molecular event
(activation of just one oncogene)
is never enough
Experiments on oncogene
cooperation
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/myc_ras(Sinn).gif
Synergistic effect
myc and ras
together
show dramatic
synergy.
myc alone
is a weak oncogene,
ras alone is somewhat stronger
an oncogene but still weak,
%
of
mice
without
tumors
IN VITRO WE SEE THE SAME
EFFECT
Fibroblasts with activated ras (top)
or activated myc (bottom) alone
do not undergo transformation.
However, co-expression
of activated ras and myc (middle)
does lead to foci of transformed cells
Ras and Myc cooperate
as they belong to different signaling
cascades
MYC pathway
RAS pathway
Cells are “durable”.
Most systems
have double and triple controls.
To break control of proliferation,
fatal errors should occur
in two or more signaling cascades
Tumor suppressor genes
Have been
theoretically predicted
by Alfred Knudson
in 1971
(Two-hit hypothesis)
http://www.djo.harvard.edu/meei/OA
Familial retinoblastoma
http://www-ermm.cbcu.cam.ac.uk/fig002frb.gif
Mutation should be in retinal cell
•http://pathology.uthscsa.edu/MSII/image1/Figure10.13.jpeg
Retinoblastoma
If 2nd mutation
arises in
connective tissue
Osteosarcoma
Two-hit hypothesis relates to TSGs
only
Oncogenes – activating mutation – damage of one allele is
enough.
Gain-of-activity mutation.
One mutation = disease.
Tumor Suppressor Genes – inactivating mutation – when one
allele is damaged, second allele stays functioning.
Loss-of-activity mutation.
One mutation = predisposition. Two mutations = disease.
Most cancer syndromes result from inherited TSG mutations
Inherited conditions that
increase risk for certain cancers
Strong or weak predisposition to cancer
development
INSTABILITY OF GENOME
as a fundamental feature
of a cancer cell
http://www.genetics.psu.edu/Faculty/photos/research/
Natural diversity of tumors
Tumors developed in the same organ
and presented with the same histology
Have the same name
(e.g. Infiltrating Moderately Differentiated
Squamous Cell Carcinoma of Lung)
But molecular picture of mutations in this tumors
can be totally different
The same effect can be obtained
by means of any genomic event.
Oncogenes
-- Activating point mutations
-- Translocation under strong promoter
-- Amplification
-- Overexpression
Tumor
suppressors
-- Inactivating point mutations
-- Promotor methylation
-- Gross chromosomal deletions
-- Underexpression
Nature of event (type of change) is random;
Choice of event (particular gene) is random;
RATES of molecular events in
cancer cells
Random gain of mutations
(low-level; natural cause)
Early stages
of natural cancer
in elderly
Forced gain of mutations
(median level; X-ray,
chemical carcinogens)
Early stages of cancer
in exposed people
Very high rate of mutations
(cell lost one or more major mechanisms of DNA repair)
Early stages of cancer in certain syndromes;
late stages of almost any cancer
Naturally occurring
mutations
How we can count mutations?
Human HPRT gene
Cells can survive without HPRT.
Cells are resistant to 6-thioguanine poison
ONLY if HPRT gene is mutated
-- Located on chromosome X
-- Encodes the enzyme hypoxanthine-guanine phosphoribosyltransferase
-- Normal function of HPRT is metabolic salvage
of the purine bases hypoxanthine and guanine
into nucleotides, inosinic acid, and guanylic acid
IN VIVO complete deficiency of HPRT activity = too much purunes
is Lesch-Nyhan Syndrome (urate crystals + self-Injuring)
Partial deficiency - nephrolithiasis,
gouty arthritis, & some neurological manifestations
HPRT ASSAY
The Hypoxanthine Phosphoribsyltransferase Assay.
6-thioguanine = Poison
6-thioguanine = OK
HPRT +/+
HPRT -/Cell
that acquired mutation
in HPRT
become resistant
to 6-thioguanine compound
We can directly count mutant
colonies and compare this
number with number of cell
seeded on plate
HPRT mutation frequency
In peripheral T cells
In 49 healthy,
non-smoking adults:
rates varied 0.25 -- 9.64 x 10(-6).
What made mutation frequencies
so different (>10 times)???
1. Polymorphisms of genes metabolizing carcinogens;
2. Polymorphisms of genes responsible for DNA repair;
3. Alcohol consumption and smoking;
4. Exposure to envinronmental carcinogens;
5. Exposure to radiation
Experimental check on hypothesis
listed above
1) CYP1A1, GSTM1 and NAT2 polymorphisms have no
influence on HPRT mutation frequencies;
2) Mismatch repair genes was also not damaged;
3) Correlation between maternal alcohol consumption during
pregnancy and results of HPRT assay on T-lymphocytes
from newborns?
Early pregnancy alcohol RR of high mutation status = 1.84
Through pregnancy alcohol RR of high mutation status = 2.99
Smoking during pregnancy
have an influence on mutational spectrum,
but not on mutation frequency
Mutat Res 1999 Dec 17;431(2):279-89
Mutations in HPRT and smoking
Yes, smoking increases mutation frequency
No, smoke does not have any influence
A comparison of mutation frequencies
in the K-ras, p53 and HPRT genes
between the normal lung tissue of smokers and non-smokers
indicates that the rate of mutation in smokers
is only ~1.6 fold higher than in non-smokers
4) Exposure to envinronmental
carcinogens
ethylene oxide ; 1,3-butadiene ; benzene
JUST marginal increase in HPRT mutability
when measured as in vivo exposure
(on plant workers populations)
Cell line-based or mice/ rat-based
HPRT assays
after direct addition of carcinogen
show strong increase in HPRT mutability
Among Hiroshima-Nagasaki survivors (43 Rad in average)
HPRT rates : 1 mut per 10-8 per base pair per generation
indistinguishable from that of Japanese controls
Chernobyl clean-up workers:
40% increase in mutation rate
in the first year after accident, ….then it declined….
Conclusion: classical HPRT assay in lymphocytes
do not support any hypothesis explaining population
varaiances in mutational load
4) Exposure to radiation
Epithelial tissues
(carcinoma progenitors)
Mut load is 0.5 -4.2 x 10(-4)
– much higher than in lymphocytes
(10- 100 times higher!)
Such rate is sufficient to account
for a large proportion of human cancers
without the need of mutator phenotype
In kidney epithelium
So, at least in kidney carcinogenesis can happens
without any additional environmental/genetic causes
COMMON TYPES
OF MUTATIONS in human cells
1. Point mutations
2. Microdeletions/microinsertions (1-3 bp)
3. Large chromosomal deletions
4. Chromosomal insertions and inversions
5. Translocations
6. Aneuploidy (extra chromosome or chromosomal loss)
Point mutations
(single nucleotide changes)
TRANSITIONS
TRANSVERSIONS
PURINEPURINE or
PYRIMIDINEPIRIMIDINE
purine  pyrimidine or
pyrimidine  purine
MOST
COMMON
Pairing is possible
due to tautomeric shifts or
ionizing that allows mispairing
Pairing is energetically infavourable,
but Pur-Pur pairs are possible (G-A)
Point mutations much more tolerable
for cell reparation system,
As they unlikely to awake apoptosis pathway.
When people talk about carcinogenic mutations,
most often they talk about point mutations.
On the other hand,
central pathogenetic event
in human leukemias and lymphomas
often is a specific translocation
Carcinoma – search for point mutation;
Lymphoma – search for translocation
RATES of molecular events in
cancer cells
Random gain of mutations
(low-level; natural cause)
Early stages
of natural cancer
in elderly
Forced gain of mutations
(median level; X-ray,
chemical carcinogens)
Early stages of cancer
in exposed people
Very high rate of mutations
(cell lost one or more major mechanisms of DNA repair)
Early stages of cancer in certain syndromes;
late stages of almost any cancer
INTRACELLULAR SYSTEMS
ERRORS
producing high-rate mutations
Can be classified in three major subtypes
TYPES OF INSTABILITY
MIN
(Microsatellite instability)
CIN
(chromosomal instability)
SIN
(single nucleotide instability)
progressive accumulation of frameshifts
in microsatellite repeats
MIN phenotype
(Microsatellite INstability)
Screening for MIN
can be performed with a panel
of 5 or more loci
(D2S123, D5S346, D17S250,
BAT25, BAT 26)
Microsatellites as a withesses
(innocent bystanders)
Microsatellites in coding
regions of cancer-involved
genes
TGFbetaRII, IGFIIR,
TCF-4, BAX,
hMSH3, hMSH6,
CHK1, and BRCA2
Poly-A and Poly-CA is especially prone; e.g. (A)6(A)7 or (CA)5(CA)4
MIN detection on BAT-panel
mononucleotide tracts BAT 25 and BAT 26
three (CA)n dinucleotide repeats (D2S123, D5S346, D17S250)
and two mononucleotide tracts (BAT 25 and BAT 26).
MIN as a DIAGNOSTIC TOOL
Shorthening of repeats in subset of cells
could be diagnosed
1) both in somatic cells (lymphocytes)
and tumor cells of HNPCC patients
(colon cancer syndrome);
2) in tumor cell of sporadic cancer patients
with MIN + phenotype
MIN+  better prognosis
for pancreatic and colon carcinomas
(better immunoreactivity of the tumor)
MIN+  Worse prognosis for germ cell tumors
(testis is immunoprivileged organ)
MIN
(Microsatellite INstability)
DNA mismatch repair proteins
Recognize and bind mismatches,
especially in microsatellite repeats
Family cancer syndromes
MSH2, MSH3, MSH6,
MLH1, MLH3,
PMS1, PMS2 genes
HNPCC colon cancer
mutations
Sporadic cancer cases
20% of sporadic colon cancers
30% of sporadic endometrial tumors
MLH1 or MSH2 promoter
Methylation
30-40% of sporadic breast carcinomas
10-15% of gastric carcinomas
Loss-of MSH/MLH gene function – Primary cause of NHPCC colon tumors;
-- Secondary event in sporadic colon tumors
SIN - instability
Recent findings in NCC (Japan)
six human breast cancer cell lines
(MCF-7, ZR-75-1, T-47D, MDA-MB-231, MDA-MB-468, BT-474),
normal human mammary epithelium (HMEC) and a colon cancer cell line
(SW480) without microsatellite instability.
http://www.ncc.go.jp/en/nccri/divisions/14carc/14carc05.html
Single nucleotide instability
Spontaneous Point Mut Rates
measured by HPRT system
are higher in cancer cell
than in normal
counterpart
CIN
(Chromosomal INstability)
Looks like
LOH
(loss of
heterozygosity)
or
Aneuploidy
LOH Detected as :
Loss of one allele of
polymorphic markers
arranged on the same
chromosome
(usually by PCR).
ANEUPLOIDY Detected as:
Loss or addition of extra copy of
chromosome
(usually by FISH or
fluocytometry)
DETECTION of LOH
TWO alleles in normal tissue
versus ONE allele in tumor tissue
Informative microsatellite
(polymorphic in this particular normal sample)
Pattern of LOH can be different in clinically similar
tumors from different patients
(but some loci have LOH more often than others)
www.genlink.wustl.edu/.../image/ figure1_radford1.gif
Black – homozygous losses;
white – hemizygous….
Overlaps of deletions in breast
carcinomas
www.medfac.leidenuniv.nl/lab-devilee/ Projects/16lohsro.gif
Two different regions of LOH
on the same chromosome are
indicative of two different
frequently deleted areas
LOH appears on a certain stage of
tumor development
http://www.bentham.org/cmm1-1/miatra/Miatra-fig3-pg159.jpg
Natural difficulties with LOH technics
Every tumor contains normal cells
(stromal cells, vasculature,
lymphocytes etc…)
1.
Normal cell have normal DNA without LOH.
They contaminate population of tumor cell,
and make results unclear.
Deletions can be
homozygous or hemizygous.
Hemizygous deletions produce LOH.
Homozygous deletions produce no PCR product,
that is a situation indistinguishable from failure of PCR reaction
2.
ANEUPLOIDY
Loss or addition
of extra copy
of chromosome.
Aneuploidy alters the dosages and expression
of thousands of normal genes
(most of them are bystanders,
not a cause of cancer)
Cancer-Aneuploidy hypothesis
P. Duesberg
Unstable
Carcinogen
produce
aneuploidy
Aneuploidy
produce
cancer cell
Stable Stable
Details of Duesberg theory
carcinogens initiate carcinogenesis
with a random aneuploidy
Aneuploid cells are error prone
as chromosome segregation
and maintenance systems
are disbalanced as a result of
unbalancing of spindle proteins,
repair enzymes,
and centrosome numbers.
did, after all,
isolate the first oncogene
by age 33
P.Duesberg,
Nobel prize winner,
Does not believe in HIV virus
How to measure aneuploidy
DNA content flow cytometry
Flow Cytometry
frozen biopsy is thawed
and placed in a solution
that ruptures the cells
leaving only the nuclei
The nuclei are stained
with a fluorescent dye
that binds to the DNA
The fluorescent dye bound
to the nuclear DNA is excited
by the light and fluoresces.
intensity or brightness of the cell's fluorescence
Is proportional to the amount of DNA in the cell
Telomeres and telomerase:
story of extra stability
Reminder: Chromosomes are comprised
of a single, uninterrupted DNA molecule
complexed with proteins
(histones and others).
Telomeres and centromere
are demarcating
the two “arms”
(p and q).
Kinetochore
microtubules
Telomeres –
Ends of linear chromosomes
Repetitive DNA sequence: TTAGGG in vertebrates
Associated with specialized proteins.
Telomeres are necessary because:
- they allow cells to distinguish chromosome ends
from broken DNA and prevents chromosomal fusions
by non-homologous end-joining (NHEJ) machinery;
- they provide a mechanism for "counting" cell
divisions as they shorten with each cell division (to be
discussed in cancer section)
- they help to establish 3D structure of the nucleus
TELOMERE – story of extra
stability
During the replication the lagging strand
requires new priming for every piece
As a consequence, every telomere
shorthen by an amount
equal to primer length
How to solve telomere maintenance problem?
It’s OK for measuring of the cell life span,
it’s not OK for maintenance of germ line cells
Telomerase
is highly expressed
in germ cells and stem cells
Somatic mouse cells express telomerase,
that is why they are easier to immortalize;
human cell - do not express telomerase.
Cancer cells express telomerase !!!
http://www.blc.arizona.edu/
marty/411/Modules/Lectures
/Figures/telomerase.GIF
Telomerase enzyme
is related
to reverse transcriptase,
as it has a small RNA template
as a part of the enzyme structure.
Telomerase structure:
Accessory proteins TEP1, p23 and hsp90
also contribute to activity of the complex
biochem.uwa.edu.au/.../B352/Lectures/ Telomerase/img001.gif
P23 and HSP90
are involved in assembly
TEP1 may facilitate
DNA binding
biochem.uwa.edu.au/.../B352/Lectures/ Telomerase/img001.gif
Awakening of telomerase
M1 crisis
M2 crisis
Different cell types have different
requirements for immortalisation
Transfection of hTERT (telomerase) gene to normal human cells
gave them an extra capacity to be passaged appr. 200 times
without transforming them to tumor cells
TRUE for human fibroblasts
and for human retinal epithelial cells
FALSE for epithelial breast cells:
They require: both TEL and del of pRB or p16
Some cell type will age
even if telomerase is re-activated
(such cells are more tumorigenesis-proof)
So, tissue-specific differences are
strong !!!
CROSS-SPECIES differences
can be even stronger!!
Everyone should remember it
when modeling cancer (or other diseases) in animals
Especially in mice,
as mice are very weird
in sense of their biology
MOUSE is very different from
human
They are smaller
They life is shorter
They telomeres are longer!!!
BUT!!!!
They have less stringent regulation
of telomerase!
mice do not have the barrier
of telomerase shortening for cell proliferation
Human do have this barrier
Mice cells are easier to immortalize
in culture compared to human counterparts
Mice are not so protected from tumors like
human beings (they no need it….)
MOUSE without a telomerase
TERC-/- mouse lack the telomerase RNA component
Tissues requiring constant renewing develop normally
(gut, skin, blood cells)
Mice are viable AND FERTILE !!!No telomerase activity!!!!
Generation number 5 (inbred)
have shorter telomeres
than those of wild-type and first-generation;
Decresead fertility and wound healing and,
as well as an increase of death in utero and
increase in failures of neural tube closure
Gonzales-Suarez et al., Nature genitics, 2000
CHEMICAL TUMORIGENESIS IN TELOMERASE DEFICIENT MICE
TERC-/- mice develop
less papillomas
in multi-stage
skin tumorigenesis;
only 37% of mice
ever develop them.
Papillomas regress
1 week after stop of
TPA treatment
TERT-/- mouse lack the catalytic
telomerase component
Looks the same.
After some generations offsprings
become prone to pre-mature aging and tumors!!!
Contradiction!!!
TERC-/- mice were checked in
multi-stage skin tumorigenesis experiments (artificial initiation of the tumors);
In this case absence of telomerase prevents immortalization of mutated cells
TERT-/- mice were checked in spontaneous tumorigenesis
which is very different from forced tumorigenesis
Why mice without telomerase are
PRONE to spontaneous tumors ?
As telomeres erode,
genomic instability is increased
due to chromosome fusion/breakage.
In mice, telomeres can be extended
by alternative ways ("ALT"),
accounting for immortalization
of telomerase-null cells.
ALT (alternative lengthening of
telomeres)
2. could also be caused by retrotransposition or recombination
ALT cell lines are characterized by the combination of
no detectable telomerase activity
and the presence of telomeres
of very heterogeneous length,
ranging from very short to much longer than normal
ALT telomere lengthening can be maintained by:
1. Another (..mutated..) type of telomerase,
that can not be recognised biochemicaly OR
What is the minimal tumor cell?
(after all)
Activation of RAS oncogene
Expression of Large T-antigen
(that inactivates RB and p53)
hTERT expression
(telomerase re-activation)
Minimal tumor phenotype
requires 3 genetic events
Creation of human tumour cells with defined genetic elements.
Hahn WC, Counter CM, …..Weinberg RA. Nature 99