The incidence of nasopharyngeal carcinoma (NPC) is very high in southern China (including Hong Kong) and in Taiwan, and in territories where individuals with ancestral origins from these regions comprise significant population constituents (such as Singapore). The aetiology of NPC has long attracted interest, with there being, historically, a major focus on environmental risk factors. NPC is one of the clinical conditions most strongly associated with EBV; all tumours contain EBV genomic DNA in episomal (latent infection) form.
In the early 1990s, Epstein-Barr virus (EBV) strains with variant latent membrane protein 1 (LMP1) gene-coding sequences began to be isolated from nasopharyngeal carcinoma
(NPC) biopsies from China and Taiwan. LMP1 isoforms expressed from these variant LMP1 genes were demonstrated to have enhanced transforming potential in laboratory assays. These findings led the authors of these studies, and others subsequently, to propose that such variant strains may be risk factors for the development of NPC, particularly because preliminary studies demonstrated that such variants were considerably more commonly recovered from NPC biopsies than from healthy virus carriers in the general population in high-incidence regions.
In this project, there has been a major focus on the study of the LMP1 isoforms B95-8 LMP1 (coded by an infectious-mononucleosis-derived EBV strain from the Western hemisphere) and CAO LMP1 (an
isoform coded by a variant EBV LMP1 gene recovered from a nude-mousepassaged NPC derived from a biopsy from a patient from the Shanghai area of China), particularly from the standpoint of probing differences in the biological activity of these isoforms from a cellular transformation standpoint.
An immortalised human fibroblast cell line (GM847), used for initial verification of correct construction of LMP1-expression plasmids, was used for further studies into the transforming capacity of the B95-8 and CAO isoforms of LMP1, because the intrinsic features of this immortalised cell line constituted a platform for the study of cellular transformation which was considered analogous to a study in which normal cells were converted to tumour cells by
the sequential addition of specific factors (and thus providing a defined platform for studying a single additional factor). The capacity for serum-independent growth of LMP1-expressing and non-LMP1-expressing cells was determined by an isotopic protocol. LMP1 expression (of both B95-8 and CAO isoforms) in the immortalised human fibroblast line rendered cells resistant to the growth-inhibiting effects of a low serum environment; B95-8 and CAO isoforms were found to be equipotent in this respect.
An expression profiling study was also completed both as a means of gaining additional understanding of the biological mechanisms by which LMP1 induces growth promoting effects, and more particularly to determine if any broad differences existed between
the gene expression profile induced by the expression of the B95-8 LMP1 isoform or the CAO LMP1 isoform in immortalised human GM847 cells. The transcript from one particular gene (AKR1C3) was determined to be transcribed at an approximately ten-fold increased level in both CAO clones analysed in the expression profiling study. This result was verified by relative quantification, real-time PCR using SYBR Green I reaction chemistry. Expression in both samples analysed, with PCR verification, makes this finding highly reliable. In addition to its role in prostaglandin metabolism, the protein coded by AKR1C3 may have a role in cellular growth and differentiation control, which could be particularly relevant as a possible additional factor to the more obvious mechanisms such as NF-KB activation already identified in other studies as effector pathways
differentially induced by CAO LMP1 in contrast to the induction levels attained by B95-8 LMP1.
The observations first made in the early 1990s that NPC-derived strains had increased biological activity have continued to be supported. However, it is now clear that such strains are widely distributed in China and are not overrepresented in NPC, and that all strains have pathogenic significance, including the B95-8 strain; so that these laboratory results may be of little clinical importance. However, despite these observations, studies into the differential transforming capacity of LMP1 isoforms coded by geographic variant viruses (particularly those from China) are ongoing, possibly because of the interest that
strain-related transformation differences continues to generate. It is possible, however, that such work of itself may identify important mechanisms in the pathogenesis of NPC.