Role of latent EBV genes in the induction of genomic instability in Burkitt’s lymphoma
Epidemiological and molecular evidence link Epstein-Barr virus (EBV) infection to a variety of lymphoid and epithelial malignancies but the contribution of the virus to tumorigenesis is unclear. Genomic instability, defined by the establishment of a mutator phenotype and characterized by the occurrence of non-clonal chromosomal aberrations, excessive DNA damage and defects in DNA repair, is the hallmark of malignant transformation. In the studies described in this thesis, we have explored the possibility that EBV may promote malignant transformation by inducing genomic instability. As a first step in this analysis, the presence of non-clonal chromosomal aberrations, including dicentric chromosomes, chromosome fragments, gaps, rings, satellite associations and double minutes, was investigated in EBV positive and negative Burkitt s lymphoma (BL) cell lines, in vitro EBV converted BLs and EBV genome-loss variants of EBV positive tumors. EBV carriage was associated with a significant increase in abnormal metaphase plates with prevalence of dicentric chromosomes, fragments and gaps. Increased phosphorylation of H2AX and lengthening of telomeres were detected in EBV positive cell lines suggesting DNA damage and telomere dysfunction as possible molecular mechanisms. Analysis of EBV gene expression revealed an increase of abnormal metaphases in cells expressing EBV latency I and further increase in latency III, suggesting that more than one viral protein may be responsible for this phenotype.
EBNA-1 is always expressed in EBV carrying proliferating cells. We investigated therefore the occurrence of genomic instability in cells expressing stable or inducible EBNA-1. Chromosomal aberrations, increased DNA damage and activation of the DNA Damage Response (DDR) as detected by phosphorylation of the DNA damage sensing kinase ATM and its downstream target histone H2AX, were observed in EBNA-1 expressing cells. These signs of genomic instability were associated with a significant increase of endogenous reactive oxygen species (ROS). Bioinformatic analysis of EBV regulated genes identified four genes within the ROS metabolic pathway as possible targets of EBV transcriptional regulation. The catalytic subunit of the ROS producing NADPH Oxidase 2 (Nox2) was shown to be selectively upregulated in EBNA-1 expressing cells. The involvement of Nox2 in the production of ROS and induction of genomic instability was confirmed by functional inactivation using chemicals and RNAi.
The possibility that more than one latency associated EBV product may be involved in the induction of genomic instability was addressed by investigating the occurrence of chromosomal aberrations increased DNA damage and DDR activation in a panel of transfected sub-lines of the B-lymphoma line BJAB carrying individual latency genes. In addition to EBNA-1, expression of EBNA-3C and LMP-1 was associated with these phenotypic markers of genomic instability. Each of these viral proteins appears to promote genomic instability through a different mechanism. Only EBNA-1 directly induced DNA damage via ROS, while expression of LMP-1 was associated with inhibition of DNA repair via downregulation of ATM, which resulted in failure to phosphorylate Chk2 and consequent inactivation of the G2 checkpoint. EBNA-3C expression induced a high degree of aneuploidy that was associated with inactivation of the mitotic spindle checkpoint and transcriptional downregulation of BubR1.
Collectively these results indicate that multiple cellular functions involved in the maintenance of genome integrity are independently targeted by EBV, pointing to the induction of genomic instability as critical event in viral oncogenesis.
List of scientific papers
I. Kamranvar SA, Gruhne B, Szeles A, Masucci MG (2007). "Epstein-Barr virus promotes genomic instability in Burkitts lymphoma." Oncogene 26(35): 5115-23. Epub 2007 Feb 26
https://pubmed.ncbi.nlm.nih.gov/17325665
II. Gruhne B, Sompallae R, Marescotti D, Kamranvar SA, Gastaldello S, Masucci MG (2009). "The Epstein-Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species." Proc Natl Acad Sci U S A 106(7): 2313-8. Epub 2009 Jan 12
https://pubmed.ncbi.nlm.nih.gov/19139406
III. Gruhne B, Masucci MG (2009). "Three Epstein-Barr virus latency proteins independently promote genomic instability by inducing DNA damage, inhibiting DNA repair and inactivating cell cycle checkpoints." (Submitted)
History
Defence date
2009-06-11Department
- Department of Cell and Molecular Biology
Publication year
2009Thesis type
- Doctoral thesis
ISBN
978-91-7409-498-5Number of supporting papers
3Language
- eng