Detection of Epstein–Barr virus infection and gene expression in human tumors by microarray analysis
Introduction
Epstein–Barr virus (EBV) is a gammaherpesvirus that infects lymphoid and epithelial cells (for a review, see Kieff and Rickinson, 2001). EBV is considered as an oncogenic virus for its transforming property that mediates the latently infected B-cells to proliferate (immortalization), and the virus possesses an oncogene BNLF1 that transforms rodent cells in vitro (Li et al., 1996). Infection caused by EBV not only results in infectious mononucleosis, but is also associated with Burkitt's lymphoma, B and T leukemia/lymphomas, nasopharyngeal carcinoma, breast cancer and gastric cancer (Kieff and Rickinson, 2001, Herrmann and Niedobitek, 2003). The virus has also been detected in many lymphoepithelioma-like carcinomas in organs or tissues that include the bladder (Gazzaniga et al., 1998), cervix (Shoji et al., 1997, Tseng et al., 1998, Sasagawa et al., 2000), gastrointestinal tract (Cho et al., 2001, Kijima et al., 2001, Hao et al., 2002, Liu et al., 2002), kidney (Creager et al., 1998), lung (Castro et al., 2001), oral cavity (Jang et al., 2001, Gonzalez-Moles et al., 2002, Sand et al., 2002, Shimakage et al., 2002, Higa et al., 2003), skin (Shirasaki et al., 2002) and thyroid (Lam et al., 1999, Tulbah et al., 1999). Interesting clinical findings have been noted on the expression of different EBV genes in odontogenic and non-odontogenic tumors (Jang et al., 2001), because these cancers occur at the locations close to that of nasopharyngeal carcinoma. The oral cavity is the location with tissue characteristics close to the nasal pharynx and yet the etiology of nasal pharyngeal carcinoma, but not oral cancers, has been associated with EBV. In Swedish populations, systematic polymerase chain reaction (PCR) screenings detected EBV in 32.1% of oral tumors that include both oral squamous cell carcinoma (37.9%) and oral lichen planus (26.1%) (Sand et al., 2002). Transcripts of EBV genes, including BYRF1, BNLF1 and BZLF1, were present in oral tumors but not in normal epithelia, suggesting the involvement of EBV in neoplastic transformation in oral cancers (Gonzalez-Moles et al., 2002, Shimakage et al., 2002, Higa et al., 2003). In gastrointestinal tract-related tumor biopsies obtained in Kyushu, Japan, 7.3% of gastric carcinomas were positive for EBV EBERs as determined by in situ hybridization (Kijima et al., 2001), whilst among the gastric tumor biopsies obtained in China, 6–9% were tested positive for the presence of EBER RNA (Hao et al., 2002). An EBV prevalence rate of 4.6% was also found in Chinese patients with colorectal cancer (Liu et al., 2002). Lastly, PCR has detected EBV DNA in lymphoepithelioma-like carcinoma of uterine cervix. Reverse transcription-PCR (RT-PCR) detected EBER mRNA in 74% of cervical carcinomas, 83% of cervical intraepithelial neoplasias, and 23% of normal cervices among Japanese patients (Sasagawa et al., 2000), implying the association of EBV in these cancers in the region. Although EBV was detected in these studies, whether EBV is present in these tumors remains unclear. This is because these studies employed PCR to detect EBV DNA, which may lead to false-positive results caused by carry-over contamination (Wiedbrauk and Stoerker, 1995), and the amplification of target genes from just few copies of EBV genome in small numbers of infiltrating lymphocytes that were present in the tumor biopsies (Cho et al., 2001). Additionally, RT-PCR normally detects the transcription of only few genes at one time (Mahony and Chernesky, 1995) and cannot be employed to examine the transcription of multiple EBV genes. In the present study, it is shown that EBV in human tumor biopsies can be detected by microarray and demonstrate the usefulness of the EBV-chips.
Section snippets
Materials, cell line and biopsies
Enzymes used in this study were purchased from Stratagene (La Jolla, CA, USA) unless otherwise specified. Chemicals and reagents were obtained mainly from Sigma (St. Louis, MO, USA). All reagents were used according to the recommendations of the manufacturers. P3HR1 is a Burkitt's lymphoma cell line that is latently infected by EBV (Chang et al., 2003). EBV in P3HR1 cells was reactivated to enter the lytic cycle by treating the cells with 3 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) and
Hybridization with mRNA purified from P3HR1 cells
To assess the EBV genes expressed during latency, biotin-labeled cDNA fragments were prepared with mRNA isolated from P3HR1 cells and then employed to hybridize the DNA probes on the EBV-chip. Hybridization results revealed that only the DNA fragments that contained the regions between nt 6019–7450 (no. 4), nt 12003–14987 (dot R or the W-repeated region) and nt 52865–54567 (no. 29) were detected (Fig. 3A). These regions contain the DNA encoding EBER, 5′ region of the EBNA transcripts, and the
Discussion
In this study, a small nylon membrane on which 71 EBV DNA fragments were produced was employed to evaluate the usefulness of microarray in detecting viral infection and gene expression in various human tumor tissues. The result showed that the EBV-chip could detect specifically the presence of EBV latent and lytic transcripts in P3HR1 cells, while the cDNA from the control cell lines, U937, yielded a negative hybridization result (Fig. 3). This result is consistent with the data obtained from a
Acknowledgements
The authors wish to express our gratitude to Dr. Hong-Chen Chen at National Chung Hsing University and Dr. Tzong-Shin Tzai at National Cheng Kung University Medical College for providing tissue specimens. This research was support partly by funds from National Health Research Institutes of ROC (NHRI-EX91-8901SL), NSC94-3112-B-182-004 to S.T.L. and Chung Shan Medical University (CSMU 92-OM-B-018) to Y.T.L. and C.L.
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