MDM2 Dependant Induction of Error Prone DNA Polymerase beta by EBV-LMP1 Oncoprotein.

Epstein Barr Virus (EBV) is able to immortalise B lymphocytes in vitro and is associated with several human tumours, such as Hodgkin’s disease (HD), suggesting that this virus could play a role in the initiation of the tumoural process. The LMP1 protein of EBV, coded by the LMP1-BNLF1 gene, was rapidly suspected to be involved in this process due to its oncogenic characteristics. LMP1 functions as a constitutively activated member of the tumour necrosis factor receptor superfamily, and activates trough its carboxyterminal cytoplasmic region several signalling pathways, including NF-kappa B. In order to identify new LMP1 target genes, we studied the transcriptome of the B lymphoid Ba/F3 cell line stably transfected with LMP1-BNLF1 genes from HD Reed-Sternberg tumour cells as well as from normal cells, using the cDNA microarray technique.

We show that the expression of several genes was modulated by the LMP1 protein, independently of the cellular origin of the LMP1 protein. Among overexpressed genes, we focused on the DNA polymerase-beta (beta-pol) gene. To confirm and extend this cDNA result, we demonstrated in different cell lines that the beta-pol protein expression was under the control of LMP1. In vivo and in vitro functional tests showed that LMP1 induced overexpression of beta-pol paralleled its increase in activity.

Regulatory sequences of the human beta-pol gene includes three motives that match the Sp1 factor binding site motive. LMP1 did not modulate Sp1 RNA neither protein expression. By contrast, we observed that MDM2 and beta-pol RNA expression were increased in parallel by LMP1, an effect trigerred by the carboxyterminal region of LMP1. Then, we demonstrated that MDM2 protein overexpression was associated with increased beta-pol protein expression.

Since MDM2 promoter is regulated by NF-kappaB, we investigated whether up-regulation of MDM2 and beta-pol were NF-kappaB pathway dependant. We show that inhibition of NF-kappaB by overexpression of an I-kappaB dominant negative down-regulated both MDM2 and beta-pol expression.

In conclusion, we demonstrated that the oncoprotein LMP1 regulates overexpression of beta-pol through NF-kappaB signaling pathway and the MDM2 protein, and that MDM2 was itself regulated by NF-kappaB. Beta-pol is the more error prone of all the known eucaryotic DNA polymerases. Elevated expression of beta-pol, an event found in many human tumours, has been shown to generate a mutator phenotype which is extensively associated with cancers. Our work leads to the hypothesis that LMP1 protein could play a role in the genetic instability in EBV associated malignant diseases such as HD through over-expression of beta-pol.