Figure 1.
High LMP1 Expression Correlates with the Development of Lymphoma
LMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice.
(A) Lymphomas are identified with a number (1–7). Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band. Actin was used as a loading control.
(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma. Scale bar, 20 μm.
Figure 2.
LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4. This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains.
(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−). Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant. This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.
(C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice. Actin was used as a loading control.
Figure 3.
LMP1 Promotes B Cell Survival and Proliferation In Vitro
(A) MTS assay of splenocytes from WT and LMP1 transgenic mice. Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d. The results are the mean ± SEM of triplicate samples averaged from multiple mice where “n” the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas.
(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d.
(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2. Percentages of cells in each quadrant are shown.
Table 1.
Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas
Figure 4.
Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture
(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes. The L32 and GAPDH housekeeping genes were used as a loading control. Arrow indicates the position of the protected probe.
(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.
(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations. Shown are the results from LMP1 transgenic lymphoma 3. The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.
Figure 5.
LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3
(A) Relative expression of IL10, IL15, and IFNγ mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay. Mouse lymphoma cell lines 967 and K46μ were used as controls. Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32. The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967.
(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1. Arrows indicate the positions of the α and β isoforms of Stat3. Actin was used as a loading control.
(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice. Scale bar, 20 μm.
Figure 6.
LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3α/β, and (B) activated p-mTOR, and total levels of FoxO1. Arrows indicate the positions of α and β isoforms of GSK3. The white line indicates that intervening lanes have been spliced out.
(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27. Actin was used as a loading control.
Figure 7.
Akt, NFκB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells
MTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes. Splenocytes were cultured with or without inhibitors of NFκB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations. The results are the mean ± SEM of triplicate samples. Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed. This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results.
Figure 8.
Analysis of Akt, NFκB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells
(A–D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFκB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFκB inhibitor, BAY11–7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations. Arrows indicate the positions of α and β isoforms of Stat3. Actin was used as a loading control.