Molecular cloning and genomic organization of the mouse AE2 anion exchanger gene.

The molecular organization of the AE2 (SLC4A2) gene, a member of the multigene family encoding sodium-independent chloride/bicarbonate anion exchangers, has previously been described in both humans and rats. In these two species, AE2 shows alternate promoter usages and tissue-specific expression of isoforms in a similar, but not identical, fashion. Here we report the molecular cloning and organization of the entire mouse AE2 gene. The gene consists of 23 exons and 22 introns and spans about 17 kb. Moreover, it drives transcription of N-terminal truncated isoforms from alternate promoter sequences in a way analogous to that described for rat and/or human orthologs. Thus, sequences within intron 2 function as overlapping alternate promoters for truncated isoforms AE2b(1) and AE2b(2), and sequences of intron 5 drive transcription of isoforms AE2c(1) and AE2c(2). Each of these variants has a specific alternative first exon, while remaining exons are common to the complete form of the message AE2a, the diversity at 5' leading to different N-termini in corresponding encoded proteins. As expected, mouse AE2 promoter sequences and the patterns of tissue expression of AE2 isoforms resemble rat counterparts more closely than human ones.

In vivo gene transfer of CD40 ligand into colon cancer cells induces local production of cytokines and chemokines, tumor eradication and protective antitumor immunity.

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 on antigen-presenting cells, is essential for the initiation of cell-mediated and humoral immune responses. In this study, we investigated the antitumor effect of in vivo gene transfer of CD40L to tumor cells using an adenoviral vector (AdCMVmCD40L) in a murine CT-26 colon cancer model. We found that injection of AdCMVmCD40L caused tumor regression in a dose-dependent manner. A complete regression of tumor was observed in 81% of mice treated with 10(9) p.f.u. of AdCMVmCD40L. The antitumor effect induced by CD40L was mediated by CD8+ T cells and was associated with the generation of tumor-specific cytolytic T lymphocytes (CTL). Animals that eradicated the tumor were protected against tumor cell rechallenge, and both CD4+ and CD8+ T cells were involved in specific protective immunity. Treatment with AdCMVmCD40L in one tumor nodule also caused complete regression of established tumors at distant sites. The antitumor effect elicited by AdCMVmCD40L was associated with the intratumoral production of IL-12 and IFN-gamma and with an increased intratumoral expression of chemokines such as MIP- 1alpha, MIP-1beta, MIP-2, RANTES, and eotaxin. These data demonstrate that intratumoral injection of AdCMVmCD40L induces a powerful cascade of chemokines and cytokines in the tumor mass and stimulates an efficient antitumor immunity leading to regression of established colon cancer and protection against tumor cell rechallenge.

Tissue-specific N-terminal isoforms from overlapping alternate promoters of the human AE2 anion exchanger gene.

Previously, we isolated the human AE2 (SLC4A2) gene, a member of the sodium-independent anion exchanger family. Rat ortholog of this gene was reported to drive alternative transcription yielding N-terminal variants of the AE2a message. We thus analyzed the human AE2 gene in this regard. Using HepG2 cells, two alternative first exons, each splicing to exon 3 in alternative transcripts, were found to be transcribed from overlapping sequences of intron 2. Exon 1b(1) corresponds to the rat variant "b" and encodes three initial residues (MTQ) in AE2b(1) isoform that replace the first 17 amino acids of AE2a protein, while the novel exon 1b(2) encodes eight initial residues (MDFLLRPQ) in AE2b(2) isoform. The relative abundance of AE2b(1) and AE2b(2) mRNAs was about 10% of AE2a mRNA each. Alternate promoter sequences have multiple potential binding motifs for liver-enriched factors, and dual-luciferase assays indicated that they possess the ability for driving transcription in transiently transfected HepG2 cells. Tissue survey showed that expression of human AE2b(1) and AE2b(2) transcripts is restricted to liver and kidney, while AE2a mRNA was encountered in all examined tissues. Our findings reveal a characteristic tissue-specific expression of two N-terminal variants of human AE2 from overlapping sequences within intron 2, one of which is a novel isoform.