Purification De La Proteine GST

ANewDiagnosticMarker forSecreted Epstein-BarrVirus ^Encoded

LMP1andBARF1Oncoproteins intheSerumandSalivaofPatients

withNasopharyngealCarcinoma

Karim Houali,1Xiaohui Wang,1Yuko Shimizu,1Djamel Djennaoui,2 John Nicholls,3 Sylvie Fiorini,1

Abdelmadjid Bouguermouh,4 and Tadamasa Ooka1

Abstract Purpose: EBV has been associatedwith nasopharyngeal carcinomas (NPC). In North Africa, the

incidence is bimodal4the first peak occurring at f20 years of age and the second peak occurring

at f50 years. Standard diagnostic tests based on immunofluorescence using anti-IgA EBV

have shown that young North African patients have a negative serology compared with older

patients.We are interested in two EBV-encoded oncoproteins, LMP1and BARF1, which have thus

far not been studied in terms of their potential as diagnostic markers for NPC. These two viral

oncoproteins have been detected in cell culture media, so we tested whether they could be

detected in the serum and saliva of patients with NPC.

Experimental Design: LMP1 and BARF1proteins were analyzed in the sera and saliva of

young patients and adult patients with NPC from North Africa and China.We then examined

whether the secreted proteins had biological activity by analyzing their mitogenic activity.

Results: Both LMP1and BARF1were present in the serum and saliva from North African and

Chinese patients with NPC. All young North African patients secreted both proteins, whereas

62% and 100% of adult patients secreted LMP1and BARF1, respectively. From animal studies,

the secreted LMP1was associated with exosome-like vesicles.These secreted EBVoncoproteins

showed a powerfulmitogenic activity in B cells.

Conclusion: Both proteins will be a good diagnostic marker for NPC whereas BARF1is a particularly

promising marker for all ages of patients with NPC.Their mitogenic activity suggests their

implication in the oncogenic development of NPC.

Nasopharyngeal carcinoma (NPC) is a human malignancy

derived from the epithelium of the nasopharyngeal cavity. It is

one of the most striking examples of a human malignancy that

is consistently associated with a virus (1–3). The EBV genome

is contained in all malignant NPC cells and it encodes viral

proteins that contribute to the malignant phenotype (4–6).

Even though infection with EBV is ubiquitous in humans, the

incidence of NPC is extremely variable, depending on the

geographic area. Whereas the incidence of NPCs in the Chinese

population peaks at f50 years of age, there are two peaks of

incidence in North Africa—one at f20 years of age and the

second at f50 years of age (6). Because of the close association

of EBV with NPC, detection of EBV anti-IgA, anti-EA, or anti-

VCA by immunofluorescence tests in serum from patients with

NPC is used in most Asian countries. However, this test is

almost always negative for young North African patients (6).

Recent data showed a successful diagnosis of NPC by molecular

serology based on EBV-encoded proteins, DNase, thymidine

kinase, and p16 VCA used as viral antigens (7–10). Virus load

in patient blood has been used as a diagnostic marker for NPC

(11, 12), but high levels have been reported in nonneoplastic

disorders, gastrointestinal malignancies, and for lymphoproliferative

disease (13, 14). We therefore need a more reliable,

simpler, and specific diagnostic test for NPC.

Several EBV genes are consistently expressed in NPC biopsies

including genes encoding the EBERs, EBNA1, LMP1, LMP2A,

BARF0, and BARF1 (4, 15–18). Among them, only LMP1 and

BARF1 were capable of inducing malignant transformation in

rodent fibroblasts (19, 20), and were thus considered as viral

oncogenes. The 21 to 56 amino acid sequence of BARF1 was

sufficient to induce malignant transformation and Bcl2

activation (21, 22). LMP1, indispensable for B cell immortalization

(23), has been reported in one series to be present in

30% to 50% of NPC biopsies (4). By contrast, a large portion

Human Cancer Biology

Authors’Affiliations: 1Laboratoire de Virologie Mole¤culaire, UMR5537, Centre

National de la Recherche Scientifique, Faculte¤ de Me¤decine Laennec, Universite¤

Lyon-1, Lyon Cedex, France; 2Service d’ORL, Hopital Mustapha, Algiers, Algeria;

3Department of Pathology, University of Hong Kong, Pok Fu Lam, Hong Kong; and

4Institut Pasteur d’Alge¤rie, Sidi-F redj, Staoueli, Algeria

Received12/14/06; revised 6/8/07; accepted 6/13/07.

Grant support: Agence National de la RechercheMIME programme, Coope¤ ration

Inter-universitaire Franco-Alge¤ rienne no. 05 MDU

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