In this analysis, only MMP3 and MMP9 were induced more than two folds by Zta. Zta Induces MMP3 Expression To confirm Zta-mediated MMP3 induction, the mRNA and protein levels of MMP3 were further examined by using quantitative RT-PCR and ELISA, respectively. and epithelial cells and contributes to pathogenesis of several lymphomas and carcinomas. Nasopharyngeal carcinoma (NPC) is an epithelial cancer endemic in DP1 southern China, southeast Asia, the Arctic, and North Africa [1]. In the endemic areas, the strong association between NPC and EBV is supported by prevalent detection of viral genomes, transcripts, and antigens in the tumor specimens [2]. Although EBV majorly adopts latent infection in NPC tumors, a small subset of the tumor cells undergo abortive lytic infection where some immediate early or early viral genes are expressed but late lytic transcripts are rarely detected [3]C[5]. Some clues suggest that EBV reactivation into the lytic cycle is linked to development or progression of NPC. Elevated antibody titers in sera against EBV lytic antigens predict a high risk of NPC [6] and are also correlated with advanced clinical stage, poor prognosis, or tumor recurrence of NPC [7]C[9]. Meanwhile, some environmental or dietary factors associated with a high incidence of NPC act as not only carcinogens but also potent inducers of the viral lytic cycle [10], [11]. Recent studies have also suggested that EBV reactivation and certain lytic proteins enhance genome instability of NPC cells [12], [13]. Another link between lytic EBV infection and NPC comes from the potential contribution of a Gliotoxin viral lytic protein Zta to NPC metastasis. Zta, also named BZLF1, is a unique member of the basic leucine-zipper (b-Zip) transcription factors and functions as an essential transactivator for the switch from EBV latency to Gliotoxin the lytic cycle [14], [15]. It forms a homodimer and binds to its target promoters through the DNA elements that are identical or similar to the binding sites for other cellular b-Zip proteins such as AP-1 or C/EBP [16]. Through the promoter binding, Zta regulates transcription of not only viral lytic genes but also some cellular genes [17]C[20]. Previous studies indicate that anti-Zta antibodies are increased in NPC patients [21] and the patients with higher titers of anti-Zta antibodies have a poorer clinical outcome owing to high incidence of tumor metastasis [9]. Notably, an immunohistochemical study shows that positive detection of Zta protein in tumor cells is correlated with advanced NPC metastasis to neck lymph nodes [4]. The potential of Zta to promote metastasis is further supported by an study showing that stable Zta expression in a keratinocyte cell line enhances cell motility and invasiveness in a collagen gel [22]. How Zta promotes cell migration and invasion is largely unknown. Two previous studies suggest that it may involve induction of matrix metalloproteinases (MMPs), a family of zinc-dependent proteolytic enzymes associated with multiple processes of cancer progression, including cell growth, migration, invasion, and angiogenesis [23], [24]. Zta upregulates MMP9 in a cervical carcinoma cell line but the biologic effects of Zta-induced MMP9 on this cell line have not been tested previously [4]. On the other hand, MMP1 is induced by Zta in a keratinocyte cell line and essential for survival of the cells growing in a collagen gel, while the contribution of MMP1 to cell migration or invasion has not been shown [22]. These two studies indicate that Zta upregulates different MMPs probably in a cell-dependent manner. However, we are not sure whether and what Zta-induced MMPs functionally contribute to cell motility or invasiveness. For two specific aims, we used a series of studies to re-examine the expression profile and biologic functions of Zta-induced MMPs in epithelial cells derived from NPC. The first aim is to test whether multiple MMPs can be Gliotoxin co-induced by Zta in NPC cells. Since promoters of some MMP genes contain similar conversion of TW01-tetER cells with a recombinant EBV expressing green fluorescence protein [31]. SCC15 is a cell line derived from oral squamous cell carcinoma [32], and AGS is a cell line derived from gastric carcinoma [33]. All the epithelial cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone, UT, USA) at 37C with 5% CO2. MMP3 activity was inhibited by treatment of cells with 10 M MMP3 inhibitor II (Calbiochem, CA, USA) for 48 h, while MMP9 activity was inhibited by treatment with 10 M MMP9 inhibitor I (Calbiochem) for 48 h. For induction of Zta expression in HONE-tetonZ cells, the cells were treated with doxycycline (1 g/ml) (Sigma, MO, USA) for 72 h. For induction of Rta expression.