An anti-apoptotic role for galectin-3 in diffuse large B-cell lymphomas

Abstract

Increased resistance to apoptosis promotes lymphomagenesis with aberrant expression of cell survival proteins such as BCL-2 and c-MYC occurring in distinct lymphoma subtypes. Galectin-3 is an anti-apoptotic protein that protects T cells, macrophages, and breast carcinoma cells from death triggered by a variety of agents. We have found high levels of galectin-3 protein expression in a subset of B-cell neoplasms including diffuse large B-cell lymphoma (DLBCL), primary effusion lymphoma (PEL), and multiple myeloma (MM), in both cell lines and patient samples. However, we failed to detect galectin-3 in Burkitt lymphoma (BL), follicular lymphoma (FL), marginal zone lymphoma (MZL), MALT lymphoma or B-small lymphocytic lymphoma (B-SLL) cell lines or patient samples. To determine whether galectin-3 expression protects B cells from apoptosis, galectin-3-negative BL cells were transfected with a galectin-3 expressing plasmid, which resulted in markedly increased resistance to anti-Fas-induced cell death. In contrast, galectin-3-positive PEL cells transfected with an amino-terminal truncated galectin-3 vector showed increased sensitivity to anti-Fas induced apoptosis. During normal B-cell development, galectin-3 expression was lowest in germinal center and plasma B cells, from which DLBCL, PEL, and MM derive, and highest in long-lived naïve and memory B cells. This pattern of expression suggests that aberrantly increased galectin-3 levels in specific B-cell populations may yield a protective advantage during transformation and/or progression of certain B-cell neoplasms.

Figure 1

Galectin-3 immunostaining of representative B-NHL. BL (A and B) and FL (C and D) tumor cells are negative for galectin-3. DLBCL (E and F) and MM (G and H) tumor cells express high levels of galectin-3. Macrophages and dendritic cells, when present within the lymphoma (D), are positive for galectin-3, serving as an internal control for staining. Hematoxylin counterstain. Original magnification: A, C, E, G, ×200; B, D, F, ×400; H, ×1000.

Figure 2

Galectin-3 protein expression in B-cell lines. A total of 23 B-NHL cell lines were examined. All 8 DLBCL examined are shown, along with representative lines for BL, PEL, and MM. The EBV status for each line is noted below the Western blot, with the lymphoma subtype listed above. No correlation between EBV status and galectin-3 expression level was observed.

Figure 3

Galectin-3 protein levels at distinct stages of B-cell development. Western blot analysis of MACS isolated B-cell populations from human tonsil probed with galectin-3 antiserum. The levels of galectin-3 expression were determined by densitometry and normalized to the densitometry-determined tubulin expression level (data not shown) for each population. Equal protein loading was confirmed with a Coomassie Brilliant Blue gel stain (data not shown). Negative and positive controls are galectin-3-negative Raji BL cells and a galectin-3 expressing Raji BL transfectant clone, respectively. Sorted phenotypes of the B-cell populations under study were naïve (CD3−/CD27−/IgD+), GC (CD10+/IgD−), memory (CD10−/CD27+/IgD−) and plasma (CD138+) B cells.

Figure 4

Galectin-3 expression in tonsil. (A to C) GC follicle showing galectin-3 immunostaining with a hematoxylin counterstain. Positive cells within the GC consist primarily of dendritic reticular cells. The arrows in C indicate scattered, non-dendritic reticular cells staining positive for galectin-3. D: Rare lymphocytes within the GC stain positive for galectin-3. Magnifications: ×100 (A), ×200 (B), ×400 (C), ×600 (D).

Figure 5

Galectin-3 protects B cells from Fas-induced apoptosis. A: Flow cytometry analysis of annexin V and propidium iodide-stained galectin-3-positive BCBL-1 PEL cells transfected with a galectin-3C-expressing or vector control construct (left four panels) and galectin-3-negative Raji BL cells transfected with a galectin-3-expressing or vector control construct (right four panels) before or after treatment with anti-Fas antibody for 24 hours. B: Summary of cell death data, shown as the percent change in apoptosis between untreated and anti-Fas-treated cells when stably transfected with galectin-3, galectin-3C, and vector control expression constructs. Data are mean ± SD of triplicate samples and is representative of three independent experiments.