A Novel Mechanism of Alternative Promoter and Splicing Regulates the Epitope Generation of Tumor Antigen CML66-L

Abstract

This report describes the difference in the epitope generation of two isoforms of self-tumor Ag CML66 and the regulation mechanism. We identified a new CML66 short isoform, termed CML66-S. The previously identified long CML66 is referred to as CML66-L. CML66-S shares the C terminus with CML66-L but has its unique N terminus. CML66-S is predominantly expressed in testis, but is also expressed in very low levels in tumor cells, whereas CML66-L is expressed in tumor cells and testis. Differential expression of CML66-L and CML66-S in tumor cells resulted from regulation at transcription, although alternative splicing also participated in the generation of the isoforms. In addition, Ab titers to a CML66-L peptide were significantly higher than that to CML66-S peptide in the sera from patients with tumors. Finally, the Abs to full-length CML66-L in the sera from patients with tumors were correlated with the Abs in the sera from these patients to CML66-L-38, which is a fusion protein with a CML66-L-specific N terminus. This suggests that the CML66-L isoform is mainly responsible for the epitope generation. Our studies have identified the alternative promoter in combination with alternative splicing as a novel mechanism for regulation of the epitope generation of a self-tumor Ag.

An Unconventional Antigen Translated by a Novel Internal Ribosome Entry Site Elicits Antitumor Humoral Immune Reactions

“…Myotrophin interacts with rel/NF-B (35,36) and converts NF-B p50-p65 heterodimers to homodimers (37). In contrast to DC6 encoded by the cDNA sense strand of tumor Ag CML66 that we previously reported (19), the coding sequence of the isolated clone was located in the mRNA-sense strand of myotrophin mRNA; however, the coding sequence was in the 3Ј-UTR (bp 3061-3237) of myotrophin mRNA (35) (Fig. 1A).…”

“…1A). Therefore, MPD6 cannot be generated by the following two mechanisms: 1) encoding by a separated exon potentially transcribed by a cryptic promoter as shown for transcription of self-tumor Ag CML66L isoform, CML66S (19), and 2) integration into the C terminus of myotrophin by exon skipping mechanism (39).…”

“…Recently, we demonstrated that alternative splicing was the dominant mechanism for increased immunogenicity of tumor Ag CML66L (19). In addition, we proposed a "stimulation-responsive splicing" model in which dominant noncanonical alternative splicing provides a structural basis for generation of untolerized self-Ag epitopes (20,21).…”

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“…Although projects may vary in format, database mining approaches follow the same set of principles ( Fig. 1): 1) Hypothesis: A clearly-presented hypothesis based on the current biomedical literature search in a given field and previous experimental data in the lab is required to carry on a database mining project as we reported Yan et al, 2004), which is similar to that of experimental projects. Of note, the database mining referred here focuses on database mining as a free standing project rather than as a part of experimental research; 2) Scope: Database mining scopes in terms of gene numbers are far more than that examined in experimental approaches.…”

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“…Todas estas alteraciones darían lugar a un potencial Ag específico de tumor. De hecho, se han detectado respuestas inmunológicas específicas contra algunos determinantes antigénicos derivados de proteínas con splicing anóma-lo en pacientes con cáncer [49][50][51][52][53][54][55][56][57][58] .…”