Week 3 Flashcards
Characteristics of viruses
Enormous variety of structure and complexity
Comprise: genetic material (DNA or RNA) protein capsid and membrane envelope
Cannot reproduce independently of host cell, obligate parasites
Host cell functions required for:
-translation of viral mRNA (all viruses)
-genome transcription. —— depending on complexity of virus
-genome replication
Virus replication normally results in cell death
Cancer cell transformation is not a normal stage/phase of the virus life cycle
Cancer accumulation of genetic mutations
Contributions from:
Genetics, virus, diet, chemical exposure
Normal cells have a strong balance of expression of proto oncogenes and tumour suppressor genes
In cancer this balance becomes dysregulated such that oncogenes become inappropriately activated and tumour suppressor genes become inactive
Viruses that cause cancer drive this process
Biological agents classified as carcinogenic to humans
IARC: international agency for research on cancer, WHO foundation
Viruses: Epstein Barr virus, kaposi sarcoma associated herpes virus, hepatitis c, hepatitis b, human T cell lymphotrophic virus type 1, high risk human papilloma virus types hpv16, hpv18, Merkel cell polyomavirus, hiv-1
Bacteria: helicobacter pylori
Liver flukes: schistosoma haematobium, opisthorchis viverrini, clonorchis sinensis
Cancer causing viruses- diverse tumour types
Epstein Barr virus: Nasopharyngealcarcinoma,Burkitt’slymphoma, immune‐suppressionrelatednon‐Hodgkin
lymphoma,extranodalNK/Tcelllymphoma, Hodgkin’slymphoma,gastriccarcinoma
Kaposi sarcoma associated herpes virus: Kaposisarcoma,primaryeffusionlymphoma
Hepatitis b and c: hepatocellular carcinoma
HumanT‐celllymphotrophicvirus type 1: AdultT‐cellleukaemiaandlymphoma
High‐riskHumanpapillomavirustypes hpv16,18,31: Anogenitalcancers‐cervix,vulva,vagina,
penis,anus, Oropharyngealtract–tonsil,baseoftongue
Merkel cell polyomavirus: Merkel cell carcinoma (skin)
HIV1: non Hodgkin lymphoma, Hodgkin lymphoma, cervical and anal cancers, conjunctiva
Direct vs indirect carcinogens
Direct: viral oncogenes directly contribute to cancer cell transformation eg HPV, EBV, KSHV
Indirect: viruses that cause cancer through chronic infections, inflammation, and immunosuppresion- lead to carcinogenic mutations in the host eg HIV1 and immune surppression. Beta-hpv types and non melanoma skin cancer- block apoptosis of UV damaged skin cells
Some viruses difficult to place in either group: HCV, HBV, HTLV1
HIV1 and cancer
Indirect carcinogen
Profound immunosuppression in HIV 1 infected patients
Opportunistic infections-persistence of infectious agents including the cancer causing viruses
Persistent immune activation by HIV1 may lead to chronic tissue damage
HIV 1 may also contribute to cell cycle deregulation or alter the microenvironment
In HIV1 infected individuals there’s an increased incidence of cancers caused by other infectious agents including viruses
Kochs postulates
For identifying an infectious agent as the cause of a specific disease
-the infectious agent is regularly found in the lesions of the disease
-the infectious agent can be isolated in cultures from fluids or tissues of an organism with the disease
-inoculation of this culture into a susceptible host produces the same disease
-the disease can be indefinitely transmitted by the recovered infectious agent
These fail when applied to infectious agents and cancer
Causality
This is difficult to establish because virus associated human cancers:
-long latency period between primary infection and tumour development
-only small % of virus infected individuals develop the tumour
-complex multi step pathogenesis: cofactors, viral genome can be disrupted in the cancer
Virus infection is one link in a chain
No experimental animal models for the human cancer
Common persistent infections
Herpes virus Epstein Barr virus EBV:
-usually asymptomatic infection during childhood (infectious mononucleosis)
-lifelong silent (latent) infection
-95% of worldwide population infected
-only small % infected individuals develop cancer, Burkitt lymphoma
-infection alone is not sufficient for cancer development
Other factors involved- Multifactorial:
-Burkitt lymphoma- malaria, MYC oncogene chromosome translocation to IgG enhancer- c-MYC deregulation
-nasopharyngeal cancer- eating salted fish (nitrosamines); genetic changes
Epidemiology- kaposi sarcoma
Classic KS (rare)- elderly Mediterranean men
In HIV infection kaposi sarcoma is:
-20000x more common than in general population
-300x more common than in other immunosuppressed groups
Patrick Moore and Yuan Chang 1994 isolated HHV8/KSHV viral sequences from AIDs-related kaposi sarcoma tumours
-representational difference analysis RDA- sequences present in tumours and not in normal tissue
Disease association HPV
HPV is a necessary cause of cervical cancer- 99.7%
Cancer causing types: HPV 16, HPV 18, >75% of cervical cancer, >50% vaginal and vulvar cancer
Non cancer causing types: HPV 6, HPV 11, >90% anogenital warts
Cervical cancer is not hereditary
Human papillomavirus HPV and cancer
Skin cancer
Cervical cancer
Head and neck cancer on the increase in boys
HPV is a direct carcinogen: viral oncogenes contribute directly to cancer cell transformation: -E6 and E7 are viral oncoproteins
HPV- a small DNA virus dependent on the host cell for replication of the viral DNA
HPV life cycle is tightly linked to epithelial differentiation
Productive phase in suprabasal layer: vision assembly, capsid expression, viral DNA, amplification
Infect basal cells, HPV infects mitotically active cells in basal layer but undergoes productive replication in mitotically inactive suprabasal cells
HPV is dependent on the host for provision of replication factors
HPV orchestrates the host cell to induce proliferation and promote cell survival
E7 and E6 expressed by HPV virus
E7 binds and inactivates Rb causes cell proliferation, RB very important in preventing cell cycle entry and E7 gets rid of it
This would normally result in lots stress on cells this would activate P53 which would induce apoptosis in those cells
But E6 binds and degrades p53 so cells divide more rapidly
G1/S checkpoint
Several growth factors can activate cyclin D CDK4,6 complex
Which then phosphorylates Rb which results in release of TF family called E2F which drives cell cycle entry
Positive growth signals activate cyclin D/CDK4,6, complex this prevents Rb binding to E2F releasing them to activate transcription specific genes
Active E2F enhances transcription genes required for S phase progression
If cell is stressed or experiencing negative growth signals p16, p21 are activated with inhibit cyclin D/CDK4,6 complex to prevent Rb phosphorylation and arrest cell at the G1 restriction point
E7 bypasses arrest and stimulates G1/S progression, Rb pocket region- LXCXE CR2 motif, conserved in oncoproteins E1A, SV40 large T antigen
E6 blocks apoptosis
Through degradation p53
In a normal cell p53 activated by phosphorylation following DNA damage
E6AP ubiquitin E3 ligase- proteasome
Cells continue to proliferate
Hepatitis B virus vaccine
HBV endemic in china, Indonesia, Nigeria, Taiwan, and also high in rest of Asia and Africa- similar to distribution of HCC
HBV vaccine constitutes purified hepatitis B virus surface antigen HBsAg licensed in US in 1979
In US successful at reducing HBV infection once at risk population targeted
Introduced into Taiwan in 1984 all new born babies vaccinated (mother to child transmission)
NHS cervical screening programme
Cervical cytology initiated in 1940s
1988 saw emergence of a “true” programme
Commences at 25 years every 3 years until 50, 5 years until 64
Registration in central computer database known as the ‘Exeter’ system
Interlinked “failsafe” processes
HPV DNA testing now an important part of the screening programme
HPV and cervical screening programme
Cytological abnormalities in the cervix monitored by the “cervical smear” or PAP test
Squamocolumnar junction epithelium changes here so very vulnerable to HPV, epithelium thin and easy to damage
Prophylactic HPV vaccine
Virus like particles formed by HPV L1 protein
72 capsomeres L1 major capsid protein
HPV16 L1 protein expressed in insect cells (early 1990s)
-assembled into empty particles
-virus like particles VLP
-basis for the HPV vaccine- induces neutralising antibodies
How do the vaccines work- block infection (prophylactic)
Given before people become sexually active
HPV vaccines stimulate production of neutralising antibodies
Produces IgH in mucosa bind to virus before it gets to basal cells
Tumour suppressor genes: an introduction
The protein products of TSGs function to negatively regulate cell growth and/or are components of cell cycle checkpoints that ensure genomic integrity in response to genotoxic stress, regulate apoptotic pathways
Loss of TSG product function (through gene mutation) means loss of growth suppressive function and/or loss of cell cycle checkpoint control. Results in enhanced and dysregulated proliferation
Are recessive in cancer. Both alleles (inherited from mother and father) of a TSG must be eliminated or inactivated to abolish tumour suppressor properties. Are heritable if mutation occurs in the germline; are somatic if mutation occurs only in the cell where the tumour originates
Initially described by Alfred Knudson to explain the inheritance of childhood retinoblastoma: knudsons two hit hypothesis
Inherited form- one mutation in germline, second mutation somatic
Non hereditary- both mutations are somatic
Cancer genetics: familial v somatic retinoblastoma
Familial: already mutant Rb allele, first somatic mutation-> 2 mutant Rb gene copies. Early onset and bilateral
Somatic/sporadic: first somatic mutation-> mutant Rb allele, secondary somatic mutation-> 2 mutant Rb gene copies. Late onset and unilateral
Familial retinoblastoma v somatic retinoblastoma
Inherited: multiple tumours, bilateral, early onset. Increased cancer risk: soft tissue sarcomas, osteogenic sarcomas
Somatic: single tumours, unilateral, later onset. Increased cancer risk: some sarcomas
Retinoblastoma
Cancer of the retina
Thickening of optic nerve due to extension of tumour- metastatic
Childhood retinoblastoma
Childhood retinoblastoma: white light reflection, squint
Incidence/diagnosis:
-40-50 children per year in Uk diagnosed with Retinoblastoma (40% familial)
-bilateral diagnosed within first year (familial)
-unilateral diagnosis between 24 and 30 months (familial 15% and somatic 85%)
-<5% diagnosed after the age of 5
Treatment dependent on stage of diagnosis:
-lasertherapy, chemotherapy, cryotherapy, surgery, thermotherapy, radiotherapy
Over 90% cured
Familial retinoblastoma
Large deletions (about 20%)
Single base substitutions (about 50%)
Small length mutations (about 30%)
Most mutations are associated with almost complete penetrance (autosomal dominant)
Rare alleles show incomplete penetrance and reduced expressivity (low penetrance retinoblastoma)
Somatic retinoblastoma
Gene deletions
Base substitutions
Small length mutations
Familial and somatic
2nd independent mutation (~30%); hypermethylation of the 5’ region- Rb1 gene
Loss of heterozygosity (~70%):
-mitotic recombination; mitotic non-disjunction; large deletions
Autosomal dominance
TSGs are recessive at the genetic level; both alleles need to be inactivated
TSGs (eg RB1) are often dominant at the cellular level
Autosome indicates that the gene locus is on a non sex chromosome
Ie if you inherit one mutant allele, you have a high probability during your lifetime of acquiring a second inactivating mutation in that gene usually through LOH 2nd independent mutation or promoter methylation
Complete penetrance- 2nd allele always inactivated
Incomplete penetrance- lower probability of acquiring second mutation
Loss of heterozygosity at retinoblastoma locus
Heterozygosity at Rb locus (one mutant allele)— S phase chromosome replication—> mutant allele on both strands chromosome but only on one.—G2 and M phases of cell cycle, mitotic recombination—>mutant allele one strand on each chromosome.
Segregation of chromatids at end of mitosis:
-retention of Rb heterozygosity in daughter cells one mutant allele on one strand
-or loss of Rb heterozygosity in daughter cells: one daughter cell no mutant allele, one cell lacking any functional Rb gene copies
Role of the retinoblastoma protein in cell cycle control
Nomenclature: retinoblastoma gene: Rb1, retinoblastoma gene product (protein): pRB
PRB: hypo phosphorylated (one phosphate) before restriction point in cell cycle
When pRB hyper phosphorylated (3 P) passes restriction point
Cyclin/CDK activities drive cell cycle
PRB functions as a transcriptional repressor during the cell cycle
-transcriptional repression (G0, G1)
-phosphorylation of pRB (early G1) by Cyc D/CDK4/6, activates chromatin passes restriction point
-phosphorylation of pRB (late G1) drives transcription
PRB inactivation in human cancer leads to bypass of restriction point and entry into S phase
PRB function in normal cells: E2F/DP TFs regulated by pRB- regulated entry into S phase
Loss of pRB function in cancer: E2F/DP TFs constitutively active bypass of restriction point- uncontrolled entry into S phase
Regulation of pRB by the p16ink4a Cdk inhibitor
Normally cyc D/cdk4/6 phosphorylate pRB-> G1/S progression
Cellular stress ^^ p16ink4a on the cyc D/CDK4/6 dont phosphorylate pRB-> growth arrest/ cellular senescence
P16ink4a is a TSG product inactivated in human cancer
-mutation (eg familial melanoma)
-epigenetic silencing (promoter methylation)
TP53 and human tumourigenesis
Nomenclature: gene: TP53, gene product (protein): p53
‘Guardian of the genome’- protects against genetic instability
Between 30 and 50% of all human cancers carry a Missense mutations in the TP53 gene that give rise to mutant p53 protein
Suggested that p53 pathway inactivated in most (over 90%) of all human cancers ie p53 function is inhibited although TP53 it is not mutated ( the p53 pathway is inactivated in retinoblastoma)
TP53 germline mutations: role for p53 in Li-fraumeni and Li-fraumeni-like syndromes
Clinically and genetically heterogenous inherited cancer syndrome
Autosomal dominant
Early onset of tumours, multiple primary tumours within an individual multiple affected family members
Multiple tumour types:
-soft tissue sarcomas and osteosarcomas
-breast cancer
-brain tumours
-leukaemias
-adrenocortical carcinoma
~400 families world wide
TP53 is typically inactivated during the late stages of cancer progression
TP53 and colon cancer:
Normal epithelium —loss of APC—> hyperplastic epithelium —DNA hypomethylation-> early-(activation of k-Ras) -intermediate-(loss of 18q TSG)-late adenomas — loss of P53–> carcinomas—> invasion and metastasis
Function and tumour suppressor properties of p53
P53 protein is a sequence-specific transcription factor
-pro apoptotic activity
-growth arresting activity
-induces cellular senescence
-inhibits angiogenesis
Mutant p53 loses ability to activate transcription
Ie mutant p53 loses growth suppressive properties
Gain of function mutants:
-have oncogenic potential and promote genetic instability
-also increase drug resistance
How does the p53 tumour suppressor protein function
P53 tetramer activates gene transcription
P53 binds to response element (ability to bind due to p53 binding proteins and p53 translation modifications)
CBP/p300 binds to P53 and TBP (bound to TATA box)
Drives transcription
Decomeric repeat
How is p53 activated
P53 exists normally as an inactive form in non stressed cells
Cellular stress:
-DNA damage
-oncogene activation (Ras)
-hypoxia (Ras)
-loss of adhesion
-ribosomal
-reactive oxygen species
How do cellular stresses regulate p53 activity
DNA damage, hypoxia, adhesion, oncogenes, ribosomal, ROS
—> mediators: ATM CHK2. P19ARF
ATM CHK2 (tumour suppressor gene products)
Phosphorylate p53
P53:
-activates mdm2 protein which is a negative regulator p53
—p19ARF inhibits with mdm2
-active p53: effectors (CBP, TRAF, PCAF, P300, ASPP1)
Requirement for p53 regulated genes in cellular response to stress
Active p53
Transducers:
-maspin, GD-AIF, tsp1, Bal-I: anti angiogenesis
-p21, 14-3-3a, GADD45: growth arrest
-p53 R2, p48, GADD45, XPC: DAN repair
-DR5, pig3, AIP1, Noxa, Bax, puma, fas: apoptosis
Role of p53 in G1-S checkpoint control
ATM phosphorylates p53
ATM activates chk2 which activates p53 too
P53 activates p21 (Cdk inhibitor)
P21 inhibits cyclin D-cdk4/6, cyclin E-cdk2
Stops progression from G1 to S phase
P53 induces apoptosis through both transcriptional and non transcriptional mechanisms
Cellular stress (eg DNA damage)
—p53 stabilisation and accumulation -> regulation of BCL2 proteins by post transcriptional mechanisms (eg binding to BCL2 BLC-Xl)—> MOMP cytochrome c release-> acrosome formation caspase activation -> cell death
-p53 stabilisation and accumulation-> expression of pro apoptotic molecules (eg PUMA, BAX)—> regulation of BCL2 proteins , MOMP cytochrome c release
The p53-mdm2 autoregulatory feedback loop
Active p53 induces mdm2 expression
Mdm2 E3 ubiquitin ligase promotes ubiquitenation of p53 protein
Mdm2 promotes p53 poly-ubiquitylation and degradation
Drives it towards 26s proteasome degradation, inhibits p53 function
Frequency of p53 mutation in human cancer
~75% Missense substitutions, mostly (95%) in sequence specific DNA binding domain
Most p53 cancer mutations affect p53 interaction with DNA
Interaction of core DNA binding domain of p53 monomer with DNA
R248 fits minor groove of DNA
Other mutations also affect p53 association with DNA not contact residues but mutation will affect whole conformation p53
Loss of p53 transcriptional activity in cancer
Familial breast cancer susceptibility genes
BRCA1:
-predisposes to early onset breast carcinoma and ovarian tumours
-greater incidence of breast v ovarian cancer
Approx 1 in 500 women have germ line mutation in BRCA1 gene
BRCA1 accounts for about 60% of inherited breast cancer
BRCA1 mutations account for about 5% of all breast cancers and up to 12% of early onset BC
BRCA1 mutation may also be associated with some prostate cancers
Over 300 mutations have been described across whole fo BRCA1 coding region
Important founder mutation- 185delAG found in 1% Ashkenazic Jewish population
Role of BRCA1 in DNA damage checkpoint signalling
BRCA1 protein is huge protein
Ring finger ligase: BARD1 and BAP1 proteins to regulate ubiquetination of the cell site cellular proteins to regulate their activity
BRCA1 protein:
-DNA damage response- cell cycle checkpoint control, DNA repair
-transcription (MYC oncogenes)
-chromatin remodelling
Ubiquitylation
Genomic stability
Familial breast cancer susceptibility genes
BRCA2/ fanconi anaemia complementation group D1, FANCD1
Predisposes to early onset breast carcinoma and ovarian tumours
Greater incidence of breast v ovarian cancer
Increased risk of prostate cancer, gall bladder, and bile duct cancer, stomach cancer and malignant melanoma associated with BRCA2 mutations
BRCA2 mutation 6174delT found in Ashkenazic women with BC
Role for BRCA2 in DNA recombination repair
DNA damage
Resected ssDNA coated with RPA
Loading of active RAD51-BRCA2 on DNA
RAD51 nucleoprotein filament formation
Non crossover event
Cross over event
Functions of BRCA1 and BRCA2
BRCA1 and 2 are both involved in the DNA damage response
Loss of BRCA1 or 2 both results in increased sensitivity of cells to ionising radiation
The major role of BRCA2 is to modulate DNA DSB repair through its interaction with RAD51 in recombination repair
BRCA1 also has a role on recombination repair but additionally plays a wider role as a cell cycle checkpoint protein
