Advertisement

Article Figures & Data

Figures

  • Fig. 1.

    Western blot analysis of EBV gene expression in BL cell lines. Cell extracts from 2 × 105 cell equivalents were electrophoresed through 8% Tris-glycine gels, transferred onto nylon membranes, probed with specific MoAb antibodies that recognize EBNA1 (AB-1), EBNA2 (PE2), and LMP1 (S12), and developed.

  • Fig. 2.

    LMP1 expression and tumorigenicity of BL30 and BL41 cell lines. (A) Western blot analysis of LMP1 expression in the BL30 and BL41 cell lines infected with P3HR-1 or B95-8 EBV. Cell extracts from 2 × 105 cell equivalents were electrophoresed through 8% Tris-glycine gels, transferred onto nylon membranes, and probed with a murine MoAb to LMP1 (S12). (B) Growth curves of tumors induced by subcutaneous inoculation of the EBV and EBV-converted BL30 and BL41 cell lines. Each data point represents the mean tumor size of all mice in each group (5 to 10 mice per group). Mice injected with parental or P3HR1-converted cell lines were killed when the tumors had reached large sizes and impaired animal mobility. (○), Parental; (□), P3HR-1; (▵), B95-8.

  • Fig. 3.

    LMP1 expression and tumorigenicity of LMP1-transfected BL41 cells. (A) Western blot analysis of LMP1 expression in parental, B95-8-EBV–converted, and LMP1-transfected BL41 cells. Cell extracts from 2 × 105 cell equivalents were electrophoresed through 8% Tris-glycine gels, transferred onto nylon membranes, and probed with a murine MoAb to LMP1 (S12). (B) Growth curves of tumors induced by subcutaneous inoculation of LMP1 or vector-transfected BL41 cells. Growth curves of tumors from vector-transfected BL41 cells and LMP1-transfected BL41 cells are depicted for individual mice.

  • Fig. 4.

    Gross and microscopic morphology of LMP1-expressing and nonexpressing BL tumors. Female BALB/c nu/nu mice were injected subcutaneously with 1 × 107 cells from exponentially growing BL41 EBV cell line (A, B, and C); EBV-P3HR-1–converted BL41 cell line (D, E, and F); EBV-B95-8–converted BL41 cell line (G, H, and I); or LMP1-transfected (J, K, and L) BL41 cell line. Tumors were removed in toto 8 to 14 weeks after the initial inoculation and processed for histology. (A, D, G, and J) Gross morphology of Burkitt's tumors removed in toto with abstract epidermis and dermis showing in (A) and (D) mostly viable-looking tumor tissue with small areas of central necrosis, and in (G) and (J) tumors with extensive necrosis and little viable tumor (no magnification). (B, E, H, and K) Microscopic morphology (original magnification × 10) of Burkitt's tumors showing in (B) and (E) viable-looking tumor tissue, and in (H) and (I) the abrupt interface between necrotic and viable tumor tissue. (C, F, I, and L) Higher power magnification (original magnification × 40) of viable tumor tissue with patent capillaries containing red blood cells (C) and (F), and capillaries occluded with thrombi at various stages of organization (I and L).

  • Fig. 5.

    Chemokine mRNA expression in LMP1-expressing and LMP1-nonexpressing Burkitt's tumors shown by semiquantitative RT-PCR analysis. Tumor tissue was obtained from Burkitt's tumors established in BALB/c nu/nu mice by subcutaneous inoculation of 107cells from LMP1-nonexpressing (parental BL41, P3HR-1–converted BL41, or GTP-transfected BL41) or LMP1-expressing BL cells (B95-8–converted BL41, LMP1-transfected BL41, or mixtures of GTP and LMP1-transfected BL41 cells). Tumors were obtained either when tumors were greater than 8 cm2 or when they developed typical tumor necrosis and scarring lesions that are indicative of tumor regression.

Tables

  • Table 1.

    Incidence of Tumor Growth and Tumor Regression in Athymic Mice Injected With BL Cells

    Cell Line EBV Status-150Tumors/ Mice InjectedDays to Tumor Appearance-151Days to Tumor Regression-152Regressing Tumors % RegressionMaximum Size (cm2)-153
    PA682 BM2 +9/10-155 28 ± 9.5 52 ± 9.2 8/8 1004.5 ± 2.2
    PA682 PB + 9/9-155 19 ± 6.219 ± 16 6/7 86 2.1 ± 2.1
    PA682 PE2 +8/10-155 7.1 ± 0.9 28 ± 17 7/7 1001.4 ± 1.1
    JLP EBV+ + 5/1411 ± 3.3 46 ± 15 5/5 100 1.8 ± 1.4
    KK124 + 8/12 16 ± 9.1 32 ± 13 4/850 2.0 ± 1.0
    Raji + 5/5 21 ± 0.077 ± 43 3/5 60 5.1 ± 3.0
    Namalwa +14/16 14 ± 6.0 55 1/14 7.16.8
    Daudi + 7/7 8.6 ± 5.00/7 0
    ST488 3/938 ± 8.1 0/3 0
    Louckes 6/11 65 ± 26 113 1/617 9.5
    BL41 15/16 41 ± 340/15 0
    Ramos 3/429 ± 0.0 0/3 0
    CA4612/15 11 ± 6.7 0/12 0
    BL30 10/10 18 ± 6.30/10 0
    BML 895 3/10 62 ± 0.0 75 ± 44 2/3 663.6 ± 1.3
    JD38PB 8/8 13 ± 6.40/8 0
    JD38BM 6/1035 ± 14 0/6 0
    • Six- to eight-week-old female BALB/c nu/nu mice maintained in pathogen-limited conditions were exposed (total body) to 4 Gy gamma irradiation, and 24 hours later injected subcutaneously with 1 × 107 viable cells from the indicated exponentially growing BL cell lines. Animals were observed weekly for at least 140 days, and tumor size estimated in square centimeters as the product of two-dimensional caliper measurements (longest perpendicular length and width). A tumor was defined as a mass measuring at least 0.25 cm2 in surface area increasing in size for at least 2 subsequent weeks. Tumors were considered in regression either when three consecutive tumor measurements separated by 1 week showed a size reduction of at least 20% or when, in the absence of measurable size increase, tumor tissue necrosis and/or fibrosis involved greater than 25% of tumor tissue sections through the maximum diameter.

    • F0-150 EBV status was determined by RT-PCR for EBNA1.

    • F0-151 Calculated from the day of cell line injection; expressed as mean (±SD) for the group.

    • F0-152 Days to tumor regression were calculated from the day of tumor appearance to the first day regression was observed.

    • F0-153 Maximum tumor size was calculated only for tumors that underwent regression, and is expressed as mean (±SD) for the group. Progressively growing tumors were greater than 7.0 cm2 in size before mice were killed.

    • F0-155 Four mice from these groups were killed for in vitro studies before regression could be evaluated.

  • Table 2.

    Incidence of Tumor Growth and Tumor Regression in Athymic Mice Injected With CA46 BL Cells Alone or in Conjunction With EBV+ or EBV BL Cells

    Cell Line EBV Status Tumors/ Mice InjectedDays to Tumor Appearance Days to Tumor Regression Regressing Tumors % RegressionMaximum Size (cm2)
    CA46 4/48.0 ± 3.4 0/4 0
    CA46 + PA682 BM2+ 4/4 10.0 ± 3.5 20 ± 5.0 2/450 3.9 ± 1.3
    CA46 + PA682 PB + 3/39.0 ± 0.0 37 ± 9.9 2/3 66 5.2 ± 2.9
    CA46 + PA682 PE2 + 3/3 9.0 ± 0.045 ± 15  3/3 100 2.5 ± 1.1
    CA46 + EW365/5 11 ± 0.0 0/5 0
    CA46 + BL41 2/2 12 ± 1.4 0/2 0
    • BALB/C nu/nu mice were injected subcutaneously with CA46 (107 cells in 0.2 mL RPMI), either alone or mixed with 107 cells from one of five Burkitt's lymphoma cell lines (PA682 BM2, PA682 PB, PA682 PE2, EW36, or BL41). Experimental conditions and evaluation are described in the legend to Table 1.

  • Table 3.

    Incidence of Tumor Growth and Regression in Athymic Mice Injected With EBV-Converted BL30 and BL41 Cell Lines

    Cell Line EBV Status Tumors/ Mice InjectedDays to Tumor Appearance Days to Tumor Regression Regressing Tumors % RegressionMaximum Size (cm2)
    BL30
     Parental10/10 18 ± 6.3 0/10 0.0
     P3HR-1 + 5/5 16 ± 0.0 0/5 0.0
     B95-8 + 5/5 30 ± 1173 ± 15 5/5 100 3.1 ± 1.3
     Parental + B95-8 −/+ 5/5 20 ± 0.061 ± 10 4/5 80 4.3 ± 0.6
    BL41
     Parental 8/8 11 ± 4.7 0/80.0
     P3HR-1 + 10/10 17 ± 8.00/10 0.0
     B95-8 + 8/934 ± 17 68 ± 36 7/8 87 5.4 ± 2.6
     Parental + B95-8 −/+ 5/5 11 ± 5.852 ± 0.0 2/5 40 6.5 ± 3.3
    • BALB/c nu/nu mice were injected subcutaneously with 107 cells (in 0.2 mL RPMI 1640), from the indicated exponentially growing parental P3HR-1–converted or B95-8–converted BL30 or BL41 cell lines. Groups of mice were also injected with a mixture of parental EBV BL cells (BL30 or BL41 cell lines, 107 cells) and the EBV-B95-8–converted BL cells (BL30-B95-8 and BL41-B95-8, 107 cells). Experimental conditions and evaluation are described in the legend to Table 1.

  • Table 4.

    Incidence of Tumor Growth and Regression in Athymic Mice Injected With an LMP1-Transfected BL41 Cell Line

    BL41 Cell Line LMP1 Status Tumors/ Mice Injected Days to Tumor Appearance Days to Tumor Regression Regressing Tumors % RegressionMaximum Size (cm2)
    Parental 8/8 11 ± 4.7 0/8  0
    GTP 6/6 11 ± 7.1 57 1/6 17 7.2
    LMP1 +6/6 20 ± 6.9 72 ± 24 4/6 674.4 ± 1.8
    GTP + LMP1 −/+ 6/6 9.0 ± 3.350 ± 0.0 3/6 50 8.2 ± 0.9
    • BL41 cells, vector only–transfected and LMP1-transfected BL41 cells (107) were inoculated subcutaneously into athymic BALB/c mice. A group of six mice was injected in the same subcutaneous site with equal numbers (107) of vector-transfected and LMP1-transfected BL41 cells. Experimental conditions and evaluation are described in the legend to Table 1.