[Effects of two LMP1 variants on resistance of CNE1 cell strain to TGF-beta1].

Background & Objective: We had proved that different latent membrane protein 1 (LMP1) variants of Epstein-Barr virus (EBV) had different effects upon growth characteristics of a human well-differentiated nasopharyngeal carcinoma (NPC) cell strain CNE1. This study was designed to investigate the possible effects of different LMP1 variants on resistance of CNE1 to TGFbeta1 and the related mechanism(s) so as to further elucidate the intrinsic mechanisms of their growth-promoting effects upon CNE1.

Methods: The plasmids including J124 (served as a control plasmid),124-B95-8 (carried LMP1 gene cloned from B95-8 lymphocytes,B95-8-LMP1), and J124- CAO-5 (carried LMP1 gene cloned from NPC tissues,CAO-LMP1) were introduced into CNE1 by liposomal transfection. The transfected cell strains were named CNE1-V,CNE1-B,and CNE1-C,respectively. Gene and protein expression of LMP1 in CNE1 were identified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis,respectively. Then growth inhibition assay using MTT colorimetric method and flow cytometry were conducted to investigate different effects of the two LMP1 variants on resistance of CNE1 to TGFbeta1. Meanwhile,TGFbetaRI,TGFbetaRII, and cyclin D1 were detected by Western blot analysis and p15 mRNA was examined by semi-quantitative RT-PCR.

Results: Two transfected cell strains (CNE1-B and CNE1-C) stably expressing different LMP1 variants were established successfully. When the treating concentration of TGFbeta1 was 5 ng/ml, the growth inhibitory rates of CNE1, CNE1-V,CNE1-B,and CNE1-C were 31.8%, 27.9%, 10.94%, and -4.26%, respectively, and the proliferation index (PI) were (24.55+/-2.55)%, (25.43+/-2.18)%, (46.78+/-2.56)%, and (54.70+/-3.84)%, respectively. CAO-LMP1 induced complete TGFbeta1 resistance in CNE1, whereas B95-8-LMP1 induced partial resistance. Neither of the two LMP1 variants had effects on TGFbetaRI and TGFbetaRII protein expression in CEN1,whereas both of them induced cyclin D1 expression significantly. CAO-LMP1 induced higher level of cyclin D1 than B95-8-LMP1 did (P< 0.05). B95-8-LMP1 had no significant effect on p15 mRNA expression (P >0.05), whereas CAO-LMP1 down-regulated p15 mRNA level obviously (P< 0.05).

Conclusion: B95-8-LMP1 could induce partial resistance of CNE1 to TGFbeta1 and the main mechanism was correlated with up-regulation of cyclin D1 protein, whereas CAO-LMP1 could induce complete resistance to TGFbeta1 and the mechanisms were correlated with up-regulation of cyclin D1 protein as well as down-regulation of p15 mRNA.