Functional analysis of TRRAP induction in the context of EBV infection

The next issue studied in this work was whether the TRRAP protein was a rate limiting for the growth and proliferation of EBV-infected B cells. To answer this question the TRRAP expression was knocked down in EBV immortalised B cells by the RNAi technology. The RNAi technology is sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene (Fire, 1999, Hammond et al., 2001, Tuschl, 2001). To down-regulate the TRRAP protein expression, a small interfering RNA (siRNA) expression vector-based system which produces functional siRNA molecule was used. The 721 cells were cells of choice as they are LCL which can be transfected with acceptable efficiencies. 721 cells transfected with the TRRAP specific siRNA expression vector show the reduction of the TRRAP transcript and pronounced cells death as soon as 12 hours post-transfection. This effect is accompanied with a decreased number of cells in the G1 phase of cell cycle, while the numbers of cells in S or G2/M phase were not significantly modified compared with the non-transfected

cells. However, cells transfected with TRRAP specific siRNA show a reduced DNA synthesis, indicating that the cells in the S and G2 phase are rather cells in the early stage of apoptosis before DNA fragmentation and thus are not recognised as sub-G1 cells. Herceg et al., 2001 reported on TRRAP requirement for the mitotic checkpoint and the normal cell cycle progression. Work presented here confirms that finding. Recently, it has been reported that siRNA can have non-specific effects. Sledz et al., 2003 showed the activation of the interferon system by synthetic siRNA. To test for the gene specific effect of anti-TRRAP siRNA, cells were transfected with the mutated TRRAP specific siRNA expression vector or empty vector as a control. These cells behave in a very similar manner to the non-transfected cells with negligible differences which are probably due to the fact that these cells were robustly treated during transfection and the selection process. These data clearly show that a general toxicity of vector or the activation of the interferon response due to a dsRNA can be ruled out as a cause of the biological effect described in this work.

In summary, these results indicate that TRRAP is intimately related and necessary for processes involved in proliferation and survival of LCLs.

The role of TRRAP in LCLs has not been elucidated, but according to the known facts about TRRAP there are several possible suggestions. As described above, TRRAP is a part of the HAT complexes which are the co-activators of the transcription machinery and are involved in the DNA repair process. Therefore TRRAP plays an important role in the regulation of transcription, cell cycle progression and cell viability. There is a need of TRRAP for cell cycle progression and survival of EBV-immortalised B cells since the latent EBV-driven proliferation of B cells requires constant and enormous gene activation accompanied by the enhancement of genetic instability (Gualandi et al., 2001). The effect of TRRAP knock-down in LCL resulting in cell death was not surprising as a similar effect was observed earlier in mouse embryonic fibroblasts (Herceg et al., 2001). However, the role of the elevated levels of TRRAP during B cell immortalisation by EBV remains unresolved. In contrast, the same is not needed for B cells activation by CD40L/IL-4. It would be of great interest to see whether lymphoblastoid cells would be viable if the level of TRRAP would be kept at the basal level.

Although EBV transformed LCLs are generally believed to be immortalised, a series of studies (Counter et al., 1994, reviewed in Sugimoto et al., 1999, Okubo et al., 2001) provide strong evidence that EBV infected B cells are mortal and have non-

malignant properties, except for a small proportion of cells that acquire additional genetic changes as well as constitutive telomerase activity. Telomere shorting with each cell division in the absence of telomere maintenance mechanism is suggested to function as an intrinsic clock that counts cell division and eventually causes permanent cell growth arrest in human cells (Chiu and Harley, 1997, Meyerson, 2000). Activation of telomerase is observed in ~90% of human cancers, but not in most normal somatic cells (Kim et al., 1994, Chiu and Harley, 1997, Meyerson, 2000). Biochemical and genetic studies have established an association between telomere maintenance and extended lifespan mediated through expression of human telomerase reverse transcriptase (hTERT) (Sharma et al., 2003). Telomerase is a reverse transcriptase that synthesises telomeric DNA thereby compensating for telomere loss that occurs with each replication cycle. hTERT is silent in almost all somatic cells (Greider, 1999) but is up-regulated in the vast majority of human cancer cells. c-Myc was shown to be the only transcription factor capable of interacting with the hTERT promoter and inducing its expression from the chromosomal locus (Wang et al., 1998). It was found that activation of a silent hTERT gene in exponentially growing primary human fibroblasts requires TRRAP recruitment and is accompanied by both H3 and H4 acetylation. Importantly, c-Myc mutants that lose their ability to interact with TRRAP fail to activate hTERT in primary cells (Nikiforov et al., 2002). Induction of c-Myc and TRRAP which are required for hTERT expression, and thus for maintenance of the constant telomere length, could be the prerequisite, though not sufficient, for the immortalisation of EBV infected B cells.

All studies addressing the function of TRRAP in EBV immortalised B cells were performed by transiently overexpressing TRRAP specific siRNA. In that system, high level expressing cells were selectively enriched and biologically investigated. Thus, TRRAP expression was most likely knocked-down below basal level seen in EBV- negative situation. For the future it would be more interesting to generate cellular systems, which would allow the titrating of siRNA expression and eventually testing whether TRRAP expression above basal level is required in B cells immortalized by EBV. Such cellular systems could be established by expressing TRRAP specific siRNAs as conditional systems.