Tumour viruses Flashcards
Peyton Rous 1911
he excised and centrifuged fibrosarcomas from chickens. He passed the supernatant through filters with very small pores and found that, when injecting this ‘cell-free’ fluid into chicks, the majority of chicks further developed sarcomas. The agent that caused this was eventually found to be a retrovirus, now called the Rous sarcoma virus (RSV). It was further found that it was the v-src gene (not other genes, such as gag, pol and env) that was required for the induction of cancer – an oncogene
1977 Bishop and Varmus.
They found that a cDNA probe for v-scr hybridised to closely related sequences in the DNA of normal chicken cells. They discovered that the normal cellular gene (the proto-oncogene) related to v-src. This was c-src. The term proto-oncogene was coined to describe this new gene (they were not themselves transforming unless mutated and/or overexpressed. Were awarded the Nobel Prize for this in 1989.
Tang et al (2013) Nature Communications
“sequenced whole cell RNA from 4433 tumours and 19 cancer types RNA not DNA sequenced to identify viral RNA as well as mRNA from DNA viruses. Sort data into human and non-human reads => non-human reads matched to known viral genomes (used the complete RefSeq collection of viral genomes which contains 3,590 genomes excluding bacteriophages)=> amount of viral mRNA expression quantified (by calculation of the fraction of viral reads (FCR) in the total library) Fraction of cells positive for viral genomes => 96.6% of cervical tumours express HPV, much higher than all other tumours. Other cancers highlighted – head and neck - the extent of HPV involvement in head and neck cancers was not really clear until this study.Also liver tumours = (32% HBV or HCV). This screen would have probably identified any major viral involvement in cancer. However, even this enormous library may still miss things – need to be cautious - although this is strong evidence, it is also only correlative” “Interestingly, their findings provide evidence against viral aetiology for some cancers where this has been frequently proposed in the past E.g. didn’t find EBV in invasive breast carcinoma Didn’t find CMV in glioblastoma multiforme “
Zur Hausen group 1983
Nobel Prize 2008 - for the discovery of human papilloma viruses causing cervical cancer.
- Looked for viral DNA in human cervical carcinomas – earlier papers had implicated herpes virus but this didn’t fit the epidemiology - herpes was what was generally accepted at the time (HSV2) - he found HSV2 absent from cervical tumours
- Hypothesized HPV because HPV DNA had already been found in genital warts
- Used Southern blot hybridization of DNA found in tumours => found sequences that hyrbidized with HPV11 only under non-stringent conditions – suggests similar but different (he had found this in warts but therefore not in the cancer)
- Tested this new HPV16 DNA under stringent conditions against tumours from various geographical locations
- HPV16 more prevalent in German biopsies (over half) than Kenyan/Brazilian (< half) – but found HPV 16 prevails in malignant tumours in both locations
- Because of difference in demographics, suggested another virus may be also responsible - later found this to be HPV18
Zur Hausen group 1989
wanted to identify HPV16 oncogenes By that time, the HPV DNA sequence was known. Were able to clone this sequence and introduce it into a plasmid.
- Used primary keratinocytes that grow in culture (HPV-16 only infects humans => can’t use an animal model)
- Used a kertinocyte focus formation assay - When papilloma viruses are introduced into keratinocyte culture, cells start building up on top of each other (would normally grow uniformly)
- Introduced short translation termination sequences via plasmids at different positions in the viral genome to see which reading frame is required for tumourigenesis - different protein coding regions were disrupted E6/7 found to be essential: when these weren’t present (i.e. the TTSs were in the E6/E7 ORFs) – the virus was no longer grew on top of each other
- Cav - this is not cancer, but a convenient surrogate quantification of some aspect of the cancer phenotype
Scheffner et al 1990
“previous co-immunoprecipitation work showed HPV16 E6 complexes with p53 in vitro; They showed p53 is degraded in the presence of HPV16/18 E6 (but not other HPV E6) by running gel electrophoresis and fluorescently labelling p53; ATP-dependence was demonstrated using an assay with ATPγS (non-hydrolyzable ATP) where p53 degradation was not successful. Staining with anti-ubiquitin mAbs showed that p53 degradation is associated with loss of ubiquitin. (Is it reasonable to analyse ubiquitination in the reticulocyte lysate system (a lot of diturbances)?)”
Hellner et al 2009
Using Western blot analysis in primary human foreskin keratinocytes, they found that E6 and E7 were each able to downregulate expression of the epithelial adhesion protein E-cadherin and induce N-cadherin expression– both of which are molecular hallmarks of EMT. This is only correlative
Chen et al 2007
Showed that conditioned media from human foreskin keratinocytes transduced with HPV-16 E6 and E7 proteins, as well as human cervical keratinocytes stably transfected with the intact HPV-16, were able to stimulate proliferation and migration of human microvascular endothelial cells (this was shown using in vitro proliferation and migration assays). Also showed that in mice, conditioned media from keratinocytes transduced with E6 E7 or stably transfected with intact HPV16 genome also induced angiogenesis (used a Matrigel plug assay)
Satou et al 2006
- First they knocked down HBZ by siRNA and found that this suppressed the proliferation of ATL cell that both expressed Tax and did not express Tax.
- Studied the growth of T cell lines transfected with two different mutated HBZ genes - one of these mutations blocked the synthesis of HBZ protein and the other and those that disrupted the RNA structure but still left intact protein.
- Showed that HBZ promotes T cell proliferation in its mRNA form, whereas HBZ suppressed Tax-mediated viral transcription via protein form.
- HBZ RNA was also transfected into a human T cell line - did an microarray and found that activates transcription of the E2F1 and its target genes.
- Transfected cells might not represent physiological levels
Epstein, Achong and Barr (1964)
used EM in cell lines from African Burkitt’s lymphoma patients => discovered EBV as the first human tumour virus.
Diehl 1967
This was furthered by research conducted by studies attempting to determine whether EBV could transform normal B lymphocytes. For example, Diehl et al. compared effects of an EBV-producer cell line or a non-producer BL cell line control that were co-cultivated with lymphocytes. It was found that the growth of lymphocytes was only found after co-culture with the EBV-producer cells, indicating that this effect was most likely a result of EBV infection
Wood et al 2016
- Looked at the EBNAs - these are nuclear antigens. They had been known for some years to be able to regulate host gene expression but the way they do this was elucidated in this paper.
- Looked at MYC (which has been previously associated with BL) - is known to be a TF capable of driving proliferation.
- Used ChIP-seq data to show that EBNA2 bound multiple elements both upstream and downstream of MYC - many of these regions bound had previously been described as super-enhancers - verifed in two diferent cell lines.
- Suggested that EBV therefore targets MYC enhancers.
- They then wanted to have a look at if EBNA2 affected enhancer promoter interactions - used chromosome conformation capture to verify that EBNA2 induced large-scale directional reorganisation of promoter-enhancer interactions - detected 2-3-fold increases in promoter interaction frequency in the presence of EBNA2 for specific enhancers.
- They then wanted to investigate the mechanism underlying this
- Previously, BRG1 (ATPase subunit of a chromatin remodeller) has been shown to be required for the interaction of an MYC enhancer (specifc, but not going to try to member name) with the MYC promoter.
- Authors then did CHiP-QPCR analysis to show that BRG1 was associated with specific MYC enhancers and EBNA2 increased the binding of BRG1 at these sites.
- They the investiagted whether BRG1 was required for the interaction of EBN2-bound enhancers with the MYC promoter. Found that siRNA BRG1 knockdown lead to loss of promoter interactions
Chene et al 2007
Their results suggest that P. facilparum antigens such as CIDR1alpha (on infected RBCs) can directly induce EBV reactivation during malaria infection that may increase the risk of BL development in children living in malaria endemic areas.
- First A soluble form of CIDR1α was produced as a glutathione-S-transferase (GST) fusion protein, and the GST protein alone was used as control.
- Immunofluorescence studies with anti-GST fluorescent Abs demonstrated that CIDR1α, but not the GST control, binds to the membrane of Akata cells (EBV-positive BL cell line).
- They showed that CIDRalpha stimulation lead to increased EBV viral load in Akata cells (determined by measuring LMP-1 expression by RT-qPCR).
- They attributed this to lytic cycle reactivation - used an Akata cell-line based system in whcih induction of the lytic cycle is accompanied by increased GFP expression.
- Akata-GFP cells were incubated with increasing concentrations of CIDR1α and GST for 48 h, at which time the extent of cells expressing GFP was measured by flow cytometry.
- The increased proportion of cells in lytic cycle (GFP-positive) induced by CIDR1α treatment correlated with a rise in the number of viral genomes as measured by quantitative PCR.
- Essentially suggests malaria infection has an effect on EBV host balance which could be associated with increased risk of BL.
- Would be interesting to extend this data in vivo - needs to be interpretted with caution - could be interesting to see if they interact in the spleen - probability of infected red blood cells to encounter EBV-carrying B cells is high
Feng et al 2008
Showed relationship between MCC and MPPV. First identified using digital transcriptome substraction (filtering out known human RNA sequences in order to identify potential viral transcripts). Found MCPV transcripts were present in 80% of cases.
Verhaegen et al 2015
- Knew that MCPV genome contains an early region encoding large T (LTAg) and small T (sTAg) antigens.
- Used transgenic mice with sT, and found that sT expression in late-stage mouse embryos promotes neoplastic transformation of epithelia
- activation in skin of adult mice (using a Cre inducible transgene) produces similar transformation-related changes and development of lesions resembling squamous cell carcinoma.
- Thus, they established sT as potent oncogene and potential oncogenic driver in MCC in vivo.
- It has been hard to isolate individual functions of sT and LT as most studies of MCV have used shRNAs that target both T antigens.
- Might not reflect levels of actual protein and did nott show downstream mechanism
