Title

Reversal of malignant cell growth phenotype by hydroxyurea-induced loss of Epstein-BARR virus episomes: 146

Document Type

Conference Paper

Department

Haematology/Oncology

Abstract

Background: Little consideration has been given to virus-directed therapy of EBV-associated AIDS malignancies, first, because latent herpesviruses are intractable to available antiviral chemotherapy and, second, because of the uncertainty that EBV makes any contribution to the long-term maintenance of the malignant cell phenotype. Using cell lines that reflects the phenotypic heterogeneity of AIDS-related lymphomas, we examined the effect of the ribonucleotide reductase inhibitor hydroxyurea (HU), known to accelerate loss of extrachromosomal DNA, on EBV episomal copy number and subsequent cell proliferation.
Methods: EBV-positive lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) cells were treated with low concentrations (50µM) of HU. EBV DNA content in treated versus untreated cells was measured serially by Southern blot and fluorescent in situ hybridization (FISH). Single cell clones from HU-treated BL cells, obtained via the cell sorter, were examined for growth characteristics in tumorigenicity assays.
Results: Primary B cells immortalized by EBV (LCL) ceased to proliferate in HU and could not be maintained as long-term cultures. On FISH analysis of treated LCLs, individual cells were detected that did not contain hybridization signals implying complete climination of EBV episomes. The median episome count in HU-treated versus untreated LCLs was 6 versus 21(p<0.0001, Wilcoxon 2-sample test). A control LCL, IB4, which contains only integrated (not episomal) EBV DNA, was not growth inhibited in the presence of HU, implicating episomal loss and not drug toxicity in the altered growth phenotype. In the case of the BL cell line Akata, there was a 99.5% reduction of EBV DNA by Southern blot analysis after 15 cell population doublings on HU. By contrast, there was no EBV DNA loss from untreated controls. At an individual cell level, approximately half the treated cells were EBV-negative by FISH. Of 131 single cell clones obtained from the treated population, 57 were EBV-negative. Unlike EBV-positive counterparts, Akata clones "cured" of EBV episomes were no longer able to grow in 0.1% serum, form colonies in soft agar or produce tumors in SCID mice despite retaining the characteristic (8,14) rearrangement.
Conclusion: These findings provide the first evidence for successful eradication of a latent herpesvirus from any cell population. They challenge the view that EBV is important only in the initiation of tumorigenesis, but plays no role in the long-term maintenance of the malignant cell phenotype. Together with the apparent reversal of EBV immortalization observed in primary B cells, these data suggest latent EBV is an appropriate and accessible therapeutic target and have prompted initiation of clinical trials of HU in EBV-related lymphoproliferative diseases of AIDS.

Comments

This work was published before the author joined Aga Khan University

Publication

Journal of Acquired Immune Deficiency Syndromes & Human Retrovirology

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