APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth

Abstract

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.

Figure 1

(A) Predicted amino acid sequence of human APRIL. The predicted transmembrane region (TM, boxed), the potential N-linked glycosylation site (*), and the NH₂ terminus of the recombinant sAPRIL are indicated. (B) Comparison of the extracellular protein sequence of APRIL and some members of the TNF ligand family. Identical and homologous residues are represented in black and shaded boxes, respectively. TNFa, TNF-α; LTa, LTα.

Figure 1

(A) Predicted amino acid sequence of human APRIL. The predicted transmembrane region (TM, boxed), the potential N-linked glycosylation site (*), and the NH₂ terminus of the recombinant sAPRIL are indicated. (B) Comparison of the extracellular protein sequence of APRIL and some members of the TNF ligand family. Identical and homologous residues are represented in black and shaded boxes, respectively. TNFa, TNF-α; LTa, LTα.

Figure 2

Expression of APRIL. (A) Northern blots (2 μg polyA⁺ RNA/lane) of various human tissues were probed with APRIL cDNA. (B) APRIL mRNA expression in various tumor cell lines: promyelocytic leukemia HL60; HeLa cell S3; chronic myelogenous leukemia K562; lymphoblastic leukemia Molt-4; Raji Burkitt's lymphoma; colorectal adenocarcinoma SW480; lung carcinoma A459; melanoma and G361. (C) APRIL mRNA expression in four different human tumors (T) and normal tissues (N). The 18S rRNA band shows equal loading. (D) APRIL mRNA expression in primary colon carcinoma. In situ hybridization reveals abundant APRIL message in human colon carcinoma. Colon tumor tissue sections and adjacent normal colon tissue were hybridized to antisense APRIL ³⁵S-labeled cRNA, and as control, colon tumor tissue sections were also hybridized to sense APRIL ³⁵S cRNA (negative control). Top, Dark field micrographs; bottom, the corresponding light field micrographs.

Figure 3

APRIL stimulates cell growth. (A) Dose-dependent stimulation of proliferation of Jurkat cells (human leukemic T cells), determined 24 h after the addition of sAPRIL. Controls include cells treated with FasL, TWEAK, and no ligand (Control). Left, Number of viable cells; middle, [³H]thymidine incorporation; right, kinetic analysis of the effect of APRIL on Jurkat cells. The concentrations of ligands are indicated. (B) Effect of APRIL on the proliferation rate of Raji (human Burkitt lymphoma), A20 (mouse B lymphoma), BJAB (human B lymphoma), COS (SV40-transformed monkey kidney cells), MCF-7 (human breast adenocarcinoma), HeLa (human embryonic lung) cell, and ME260 (human melanoma). (C) Influence of immunodepletion of FLAG-tagged APRIL on tumor cell growth. The proliferative effect of FLAG-tagged APRIL is neutralized by Sepharose-bound anti-FLAG antibodies, but not by anti-myc antibodies. (D) Influence of FCS concentration on APRIL-induced proliferation of Jurkat cells. Data are the means ± SEM of triplicate determinations.

Figure 3

APRIL stimulates cell growth. (A) Dose-dependent stimulation of proliferation of Jurkat cells (human leukemic T cells), determined 24 h after the addition of sAPRIL. Controls include cells treated with FasL, TWEAK, and no ligand (Control). Left, Number of viable cells; middle, [³H]thymidine incorporation; right, kinetic analysis of the effect of APRIL on Jurkat cells. The concentrations of ligands are indicated. (B) Effect of APRIL on the proliferation rate of Raji (human Burkitt lymphoma), A20 (mouse B lymphoma), BJAB (human B lymphoma), COS (SV40-transformed monkey kidney cells), MCF-7 (human breast adenocarcinoma), HeLa (human embryonic lung) cell, and ME260 (human melanoma). (C) Influence of immunodepletion of FLAG-tagged APRIL on tumor cell growth. The proliferative effect of FLAG-tagged APRIL is neutralized by Sepharose-bound anti-FLAG antibodies, but not by anti-myc antibodies. (D) Influence of FCS concentration on APRIL-induced proliferation of Jurkat cells. Data are the means ± SEM of triplicate determinations.

Figure 3

APRIL stimulates cell growth. (A) Dose-dependent stimulation of proliferation of Jurkat cells (human leukemic T cells), determined 24 h after the addition of sAPRIL. Controls include cells treated with FasL, TWEAK, and no ligand (Control). Left, Number of viable cells; middle, [³H]thymidine incorporation; right, kinetic analysis of the effect of APRIL on Jurkat cells. The concentrations of ligands are indicated. (B) Effect of APRIL on the proliferation rate of Raji (human Burkitt lymphoma), A20 (mouse B lymphoma), BJAB (human B lymphoma), COS (SV40-transformed monkey kidney cells), MCF-7 (human breast adenocarcinoma), HeLa (human embryonic lung) cell, and ME260 (human melanoma). (C) Influence of immunodepletion of FLAG-tagged APRIL on tumor cell growth. The proliferative effect of FLAG-tagged APRIL is neutralized by Sepharose-bound anti-FLAG antibodies, but not by anti-myc antibodies. (D) Influence of FCS concentration on APRIL-induced proliferation of Jurkat cells. Data are the means ± SEM of triplicate determinations.

Figure 4

APRIL accelerates tumor growth. (A) Characterization of APRIL-transfected NIH-3T3 clones. FLAG-APRIL levels of the various clones were analyzed by Western blotting using an anti-FLAG antibody. Arrow, The APRIL protein; the high molecular weight protein is detected nonspecifically. (B) APRIL-expressing NIH-3T3 clones grow faster than mock-transfected clones. (C) Increased tumor growth of APRIL-expressing NIH-3T3 clones. NIH-3T3 cells (10⁵ cells) and APRIL (NIH-AP, two different clones) transfectants (10⁵ cells) were injected subcutaneously into nude mice, and tumor growth was monitored. Data are representative of three experiments with six mice per group.