but also because a particular Hsp90 conformation required for inhibitor binding exists more frequently BMS 777607 c-Met inhibitor in tumor cells. EBNA1 is an unusual BMS 777607 c-Met inhibitor protein that is translated with extremely poor efficiency, but is highly stable once it is made. Interestingly, our results suggest that, rather than decreasing the stability of EBNA1, Hsp90 inhibitors further reduce the ability of EBNA1 to be translated. A region in EBNA1 previously shown to inhibit EBNA1 translation is required for Hsp90 inhibition of EBNA1 expression. Importantly, the toxic effect of low dose Hsp90 inhibitors in LCLs is substantially reversed following enforced expression of a mutant EBNA1 protein resistant to the Hsp90 effect.
Finally, we also show that EBV induced lymphoproliferative disease in SCID mice is strongly inhibited using a nontoxic dose of 17 AAG.
Our results suggest that Hsp90 inhibitors can be used to decrease EBNA1 CAY10505 expression in a variety of different EBV CAY10505 infected cell types and thus may prove useful for treating certain EBV induced diseases. Results Hsp90 Inhibitors Decrease EBNA1 Expression in a Variety of Cell Types. To determine whether Hsp90 inhibitors alter EBNA1 expression, various types of latently infected, EBV positive cells were treated with vehicle control orHsp90 inhibitors.
Hsp90 inhibitors decreased the expression level of EBNA1 in every EBV infected cell line examined, including two different LCLlines, two different Burkitt lymphoma lines, two different NPC lines, and a gastric carcinoma line. Treatment with 17 DMAG reduced the EBNA1 expression level to 6%to 8%of its normal expression level inLCL1,LCL2, and Mutu BL lines.
As expected, expression of the cellular protein, cdc2 ,was also decreased, whereas actin expression was not affected. The inhibitory effect of Hsp90 inhibitors on EBNA1 expression in B cell lines required several days of treatment, but was apparent in epithelial cell lines within 48 h. To determine if Hsp90 inhibitors decrease EBNA1 expression outside the context of the EBV genome, EBV negative AGS gastric carcinoma cells were transfected with an EBNA1 expression vector driven by the SV40 promoter, then treated with or without 17 AAG beginning at 4 h after transfection.
As shown in Fig. 1E, 17 AAG treatment significantly decreased expression of transfected SG5 EBNA1, whereas expression of another EBV protein, LMP1, in the same vector was increased.
Of note, we found that Hsp90 inhibitors nonspecifically decrease expression of all CMV promoter driven proteins and thus did not use CMV promoter constructs for these experiments. Hsp90 Inhibitors Can Decrease EBNA1 Expression Without Affecting EBNA1 Transcript Level. The EBNA1 transcript is derived from the Qp viral promoter in EBV Burkitt lymphomas, gastric cancers, and NPC tumors, and derived from the Cp promoter in LCLs. The level of EBNA1 mRNA in HONE/Akata cells was not significantly affected by 17 DMAG treatment, suggesting that Hsp90 inhibitors do not affect EBNA1 transcription or RNA stability in this cell type. In contrast, in cells with type III viral latency, in which EBNA1 activates its own transcription from the viral Cp promoter, 17 DMAG treatment decreased the level of EBNA1 transcripts as expected, as well other viral proteins derived from Cp such as EBNA2, although LMP1 wa