Human-specific Alu detection by PCR

DNA was extracted from tumor tissues as described in protocol. Alu PCR was conducted under the following conditions: 95°C predenaturation for 5 min, and then 30 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min. Primers (sense: 5′-CAC CTG TAA TCC CAG CAG TTT-3′, anti-sense: 5′-CGC GAT CTC GGC TCA CTG CA-3′) were used to amplify a 221 bp sequence for human-specific Alu.

In situ hybridization of EBV infection

The synthetic oligonucleotide EBER1 probe (sequence: 5′-CTC CTC CCT AGC AAA ACC CTC AGG ACG GCG-3′) [10] was end-labeled with digoxigenin by tailing with terminal transferase. The labeling reaction was set up according to the manufacturer’s protocol (Boehringer Mannheim Co., Ingelheim, Germany). In situ hybridization using EBER1 probe was carried out as follows. Briefly, the glass slides were pretreated with 2% APES. 4-μm tissue slices of the induced tumors were heated at 70°C for 1 h, and deparaffinaged in xylene, then digested with 0.5% μg/ml proteinase K at 37°C for 10 min, and washed in water. The labeled probes were diluted to a concentration of 100 ng/ml in hybridization medium [25% deionized formamide, 4 × SSC (sodium citrate, sodium chloride), 50 mmol/L NaH2PO4/Na2HPO4, 1 mmol/L EDTA, 5 × Denhart's, 1 mg/ml ssDNA], and then spotted on to the tissue slices, which were covered with a coverslip. Both DNA probe and target RNA in tissue slices were simultaneously denatured at 70°C for 8 min. Then the sections were hybridized at 37°C for 4 h or overnight. The non-specific or unbound probes were removed by two post-hybridization washes as follows: 2 × SSC, 0.1 × SSC, each for 10 min at room temperature. The slices were blocked with 2% normal goat serum at room temperature for 20 min, incubated with anti-Digoxigenin antibody at 37°C for 30 min, and then washed 3 times. NBT/NCIP was used as the chromogen. Counter-staining was done to enhance visualization with nuclear fast red.

Results

The EBV-related neoplasms in the SCID mice were solid masses. 21 out of 29 mice (72%) developed tumors in their body. By autopsy, some were round-shaped nodes (Figure 1A), with distinct boundary and no envelope; others were irregular nodes, adherent to contiguous organs. The diameters of tumors were about 0.5-3.6 cm, and the cuts displayed pale and gray-red. Moreover, loci of necrosis could be observed in the center of larger tumors. Under microscope, tumor cells diffusely arranged with rare sparse interstitial fibers and tiny vessels. The tumors were dominated by large cleaved and non-cleaved cells (Figure 1B), with a few large and diverse immunoblasts and plasmacytoid lymphocytes and a few megalocytes as well. Karyokinesis could be easily observed and nucleoli were distinct in the tumor cells. The tumors in the abdominal cavity of mice often invaded into kidney, liver, pancreas, mesentery; and those in the midiastinum invaded into striated-muscles of posterior thoracic wall. All of the pathological characters revealed that the induced-tumor was highly malignant non-Hodgkin’s lymphoma (NHL).