Role of latent EBV genes in the induction of genomic instability in Burkitt’s lymphoma
Author: Gruhne, Bettina
Date: 2009-06-11
Location: Petrénsalen, Nobels Väg 12 A
Time: 09.00
Department: Institutionen för cell- och molekylärbiologi (CMB) / Department of Cell and Molecular Biology
View/ Open:
thesis.pdf (1.789Mb)
Abstract
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.
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 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
Pubmed
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
Pubmed
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)
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
Pubmed
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
Pubmed
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)
Issue date: 2009-05-21
Rights:
Publication year: 2009
ISBN: 978-91-7409-498-5
Statistics
Total Visits
Views | |
---|---|
Role ...(legacy) | 633 |
Role ... | 109 |
Total Visits Per Month
October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | April 2024 | |
---|---|---|---|---|---|---|---|
Role ... | 1 | 1 | 1 | 0 | 1 | 0 | 0 |
File Visits
Views | |
---|---|
thesis.pdf(legacy) | 491 |
thesis.pdf | 218 |
thesis.pdf.txt(legacy) | 2 |
Top country views
Views | |
---|---|
United States | 324 |
China | 72 |
Germany | 50 |
Sweden | 50 |
Finland | 14 |
South Korea | 14 |
Russia | 10 |
Japan | 9 |
United Kingdom | 7 |
Denmark | 6 |
Top cities views
Views | |
---|---|
Sunnyvale | 34 |
Romeo | 29 |
Beijing | 26 |
Kiez | 17 |
Shenzhen | 15 |
Seoul | 14 |
Ashburn | 9 |
London | 7 |
University Park | 7 |
Ballerup | 6 |