papiloma viruses "review on e6 and e7"
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SHERKO NASERIAdvicer: Dr.Babak Behnam
Cellular AND Molecular BiologySummer2011, Tehran University of medical science
HPV ( E6,E7) : Cellular Mechanism of Interaction
Cancer of the cervix
2nd most common cancer in women worldwide
Profiles like an STD (sexually transmitted disease) because of STD-dependent development
Importance
HPV is Necessary Cause of Cervical Cancer
Human papillomavirus DNA required for development of cervical cancer
HPV DNA detected in 90-100% of cervical cancer specimens compared to 5-20% in epidemiological control specimens
Bosch et al., 2002.
All characterized strains only infect epithelial cells, specifically skin anogenital mucosa oropharyngeal mucosa
HPV is Epitheliotropic
Human Papillomavirus model*
HPV Genome E1-E7 = “Early” genes
(nonstructural) L1, L2 = Capsid genes URR = upstream regulatory
region
E6 & E7 proteins play major role in immortality & malignant transformation of infected cells
E5 has role, but not required to maintain cancer phenotype
E2 Has a versus relate with E6 AND E7Munoz et al. 2006.
NORKIN,molecular virology,2008,ASM
HPV Classification & Carcinogenic Risk
Low Risk 60, 11, 42, 43, 44
Intermediate Risk
31, 33, 35, 51, 52, 58
High Risk 16, 18, 45, 56
Over 100 HPV strains identified
Risk assessment based on transformative potential of a strain’s E proteins
Low found in benign lesions only
Intermediate found in benign lesions & invasive cancers
High usually found in carcinomas; occasionally seen in benign lesions
Furumoto et al., 2002.
HPV’s E6 & E7 proteins interact with key cell cycle proteins including pRB & p53, effectively over-riding the G1/S-phase checkpoint
Mechanism1. E7 binds & phosphorylates pRB, activating E2F
transcription factor2. DNA replication proteins of host cell are then
expressed; unchecked S-phase occurs3. E6 marks p53 for proteolytic degradation so it
cannot activate apoptosis (note: absence of p53 is not necessary for E6 to cause immortalization)
Early Genes Hijack Cell Cycle Checkpoint
E6 & E7 in Cervical Cancer Progression
Furumoto et al., 2002.
Keratinocyte differentiation retarded
Checkpoint dependence goneChromosomal instability; accumulation of
oncogenic mutationsIncreased loss of cell cycle/growth control
Cancer
Consequences of the HPV Hijack
E6 CharacterizationA protein with 158 residues Four cysteine arrays—two
potential zinc fingers and molecular weight of about 18 kDa
N-terminal and C-terminal fragments encompassing residues 7-83 and 87-158
A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of tumor suppressors
HPV-16 E6 protein from SWISS-Model Repository (P03126)
High-Risk HPV Oncoproteins: E6E6 Identified Function Investigator
(1) Cell immmortalization (2) Binding of E6-associated protein results in degradation of specific host cell proteins [p53] (3) Anti-apoptotic effect(4) Chromosomal destabilization (5) Enhancement of foreign DNA integration & mutagenicity (6) Activation of telomerase (7) Blockade of interferon functions(8) Degradation of Bak protein
(1) Band et al., 1990 (2) Werness et al., 1990 & Sheffner et al., 1993 (3) Werness et al., 1990 & Thomas, 1998 (4) White et al.,1994 (5) Kessis et al., 1996 & Havre et al., 1995 (6) Klingelhutz et al., 1996 (7) Ronco et al., 1998(8) Banks et al., 1998 & 1999
Hausen, 2000.
A ubiquitin thiolester cascade model for the HPV E6 dependent ubiquitination of p53
Ubiquitin is a 8 kDa protein of 76 amino-acid
E6BP and E6AP BindingE6 binding to cellular proteins can be
considered separately from p53 binding and degradation
for several reasons :1) E6 can bind to E6BP (ERC-55/E6BP) in the
absence of p532)BPV-1 E6 binds both E6BP and E6AP but
not p533) E6 binding to E6AP is a prerequisite for
p53 binding4) E6, E6BP and E6AP can form a ternary
complex
Stimulates expression of transcription factor HIF-1α
Both HPV E6 and E7 were able independently to enhance induction of HIF-1α upon DFO treatment. Enhancement of HIF-1α stability was not restricted to high risk HPV types, as HPV11, a low risk HPV type, mediated a similar effect(Mitsuhiro Nakamura et al,2010)
Prognostic Marker: Higher levels of HIF-1α expression in early-stage invasive cervical cancer correlated to shorter overall survival time
E6 Interact with HIF1-Alpha
In cells with normal functioning p53, HIF-1α is expressed in instances of hypoxia (as its name, hypoxia-inducible factor, implies)
HIF-1α binds & stabilizes p53 to induce apoptosis of hypoxic cells, however p53 is degraded by E6 in HPV-infected cells
Instead, HIF-1α stimulates neoangiogenesis for tumor cells, providing the vascularization necessary for cancer progression
Significance of HIF-1α Expression in Cervical Cancer
Hif-1α p53 BAX APOPTOSE
If P53 not present
Hif-1α
VEGF Neoangiogenesis
E6E7
Degradation
E6
Mdm2
Keyword:Mdm2(Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and an inhibitor of p53 transcriptional activation
Evidence of HIF-1α Overexpression in Cervical Cancer
No expression of HIF-1α in normal specimensAntibody treatment
less likely to disrupt normal cells
HIF-1α expression identified by nuclear staining/ immunohistochemistry
A
B
Invasive cervical cancer specimens exhibiting strong (A) & weak (B) HIF-1α expression
Birner et al., 2000
Candidate for Anti-angiogenesis Therapy?
Reduction of HIF-1α-induced angiogenesis may slow progression rate by cutting off oxygen & nutrient supply to tumor cells
HIF-1α mediates angiogenesis through activation of VEGF pathway Vascular endothelial
growth factor stimulates angiogenesis & release of similar factors
AntiHIF-1α or antiVEGF antibody treatment may control progression of cervical cancer
High-Risk HPV Oncoproteins: E7
E7 Identified Function Investigator(1) Cell immmortalization (2) Activation of cyclins D & E(3) Induction of apoptosis(4) Inhibition of cyclin-dependent kinase inhibitors(5) Enhancement of foreign DNA integration & mutagenicity (6) Degradation of Blk tyrosine kinase(7) Inactivation of retinoblastoma protein-related pocket proteins
(1) Munger & Phelps, 1993(2) Arroyo et al., 1993 & Zerfass et al., 1995 (3) Putthenveettil et al., 1996 (4) Jones et al., 1997 & Funk et al., 1997(5) Kessis et al., 1996 & Reznikoff et al., 1996 (6) Oda et al., 1999(7) Dyson et al., 1989, 1992.
Hausen, 2000.
E7 CharacterisationThe E7 proteins are small, acidic polypeptides
composed of approximately 100 amino acidsis its ability to bind and induce degradation of the
tumor-suppressor retinoblastoma protein (pRb) via the ubiquitin pathway
In the absent of pRb،E2f is separated from pRb and start to promote cell cycle
E7 contains two conserved regions (CR1,CR2)these two conserved regions significantly contribute
to the transforming activities of high-risk HPV E7 oncoproteins
Transcription factor Dp-1 is a protein that in humans is encoded by the TFDP1 gene
E2F factors bind to DNA as homodimers or heterodimers in association with dimerization partner DP1
E7 is PleiotropicInactivation of p21CIP-
1 & p27KIP-1 (cdk inhibitors) results in growth stimulation of infected cells
Inactivation of tumor suppressor transcription factor interferon 1 (IRF-1) through direct interaction HPV-1a E7 protein from
SWISS-Model Repository (P06465)
E7 Protein S tractureCR2 contains the LXCXE
domain (amino acids 22–26), which mediates the association with the pocket proteins, and two serines at positions 31 and 32, which are phosphorylated by casein kinase II. CR3 contains two CXXC motifs that are involved in zinc binding and in protein stabilization
IRF-1 Deactivation by E7
May explain the immune-resistance mechanism of HPV-infected cervical cancer cells
1. IRF-1 activated during exposure to viral infection, IFNs, TNFα, etc.
2. Histone deacetylase (HDAC) mediates accessibility to chromatin of IRF-1 inducible genes, such as IFN-β
3. IFN-β expression stimulates anti-proliferative effect on cell
Normal role of IRF-1 in tumor suppressor mechanism
E7 interacts with HDAC and IRF-1
E7 Zinc binding CR-3 is binding to histon deacetylase
CR3 contains two CXXC motifs that are involved in zinc binding
Blocks expression of IRF-1 inducible genes by inhibiting HDAC
Result: Cell proliferation evades immune response
Mechanism of IRF-1 Inactivation
Park et al., 2000.
Histone : Acetylation and Deacetylation
HDAC model – active domain with green
HADC caused to remove acetyl group (O=C-CH3) from an ε-N-acetyl lysine amino acid on a histone
DNA expression is regulated by acetylation and de-acetylation
If acetylation: histones displaced and DNA is accessible
If deacetilation acured DNA as is wrapped around histones
Notch1 expression would inhibit expression of HPV regulatory region (URR) & subsequent E6/E7 expression
Novel protective role against HPV-induced transformation
Notch1 Signaling Pathway in HPV-Cervical Cancer
Talora et al., 2002.
New Article in 2012
Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteinsinhibited TNF-alpha-inducible NF-kB activity in human epithelial cells cultured
NF-kB influenced immortalization of cervical cells by HPV16
inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells
New Article in 2011
We established two immortalized cell lines from human oral epithelium by transducing mutant cyclin dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase with or without dominant-negative p53 into primary-cultured normal oral gingival epithelial cells using recombinant lentivirus vectors and named them MOE (mouth-ordinary-epithelium) 1a and MOE1b,respectively.
MOE1 cells kept the characteristics of normal epithelial cells without acquiring typical features of cancer cells and they could be useful not only for the study of oral neoplasm but also for other oral diseases
Bosch et al. 2002. The causal relationship between human papillomavirus and cervical cancer. J Clin Pathol 55:244-265.
Birner et al. 2000. Overexpression of Hypoxia-inducible Factor 1α Is a Marker for Unfavorable Prognosis in Early-stage Invasive Cervical Cancer. Cancer Research 60:4693-4696
Furumoto et al. 2002. Human papillomavirus (HPV) and cervical cancer. J Medical Investigation 49:124-122.
Hausen, H. 2000. Papillomaviruses Causing Cancer: Evasion from Host-Cell Control in Early Events in Carcinogenesis. J Natl Cancer Inst 92:690–8
Munoz et al. 2006. HPV in the etiology of human cancer. Vaccine 24S3:S3/1-S3/10
Park et al. 2000. Inactivation of Interferon Regulatory Factor-1 Tumor Suppressor Protein by HPV E7 Oncoprotein. J Bio Chem 275;10:6764-6769.
Talora et al. 2002. Specific down-modulation of Notch1 signaling in cervical cancer cells is required for sustained HPV-E6/E7 expression and late steps of malignant transformation. Genes & Dev 16:2252-2263
http://emc.medicines.org.uk/emc/assets/c/html/DisplayDoc.asp?DocumentID=19016
Toshiro Kibe et al.2011, Immortalization and characterization of normal oral epithelial cells withoutusing HPV and SV40 genes, Journal of the Japanese Stomatological Society,14 mrch2011
Erik R. Vandermark et al.2011, Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization,28jan2012
References
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