Alternative splicing of the human MUC2 gene☆

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Abstract

Human colon cancers differ in amounts of MUC2 mucin synthesized. However, it is unclear whether MUC2 encodes a single protein. When clones of human colon cancer cells were assayed with antibodies against the TR2 mucin repeat or non-TR2 epitopes, differences in relative expression of MUC2 proteins suggested multiple immunoreactive forms. RT-PCR analysis detected the established 15 kbp MUC2 cDNA and a novel form (designated MUC2.1) lacking the MUC2 TR2 repeat. Sequencing of cDNA and genomic DNA indicated that MUC2.1 results from an alternate splice donor. RT-PCR with splice-junction spanning primers confirmed the expression of MUC2.1 mRNA. Anti-MUC2.1 antibody stained colon cancer cells and normal colon in a pattern different from TR2-specific antibody. The presence of MUC2.1 mucin may help us to explain previous conflicting reports that have attempted to correlate the relative abundance of MUC2 protein and/or mRNA with the biological behavior of colon cancer cells.

Section snippets

Cell lines

Human colon cancer cell line HM7 is a derivative of the colon adenocarcinoma line LS174T (ATCC CL188) [22] and has been described previously [8], [23], [24]. Cell lines HM7-F6, HM7-B10, HM7-C5, HM7-E8, HM7-E2, and HM7-E3 are subclonal derivatives of HM7 that express different relative levels of human MUC2 protein. Low passage aliquots were maintained in a 5% CO2 environment at 37 °C in Dulbecco’s modified Eagle medium (Life Technologies, Rockville, MD), with 10 % fetal bovine serum, penicillin

Variation in expression of MUC2 protein in human colon cancer cell lines

HM7 is a derivative of the LS174T colon cancer cell line and has been extensively characterized [8], [23], [24]. Previous results indicate that a predominant form of mucin produced in HM7 cells is MUC2 intestinal mucin [6]. Derivatives of the HM7 cell line (HM7-F6, HM7-B10, HM7-C5, HM7-E8, HM7-E2, and HM7-E3) were isolated by limiting dilution and assayed for relative expression with anti-MUC2TR2 (monoclonal antibody CCP58) against a non-O-glycosylated peptide sequence within the MUC2 TR2

Discussion

Inherent difficulties in the resolution of high molecular weight proteins make it difficult to conclude whether MUC2 is expressed as a single apoprotein. This uncertainty is recapitulated at the mRNA level. The majority of published Northern blot hybridization reports indicate that MUC2 mRNA resolves as either a disperse signal 3–15 kb in size [6], [12], [32] or several wide bands within the same range [5], [33]. Possible explanations for this have included alternative splicing, alternate

Acknowledgements

This work was supported by National Cancer Institute Grant R01CA69480. The authors acknowledge Neil W. Toribara for providing unpublished MUC2 DNA sequences and thank Chris Yunker for excellent technical assistance.

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    ☆

    The nucleotide sequences described herein have been deposited in the GenBank database under Accession Nos. AF257165, AF257166, and AF257167.

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