Gastroenterology

Gastroenterology

Volume 123, Issue 1, July 2002, Pages 152-162
Gastroenterology

Basic–Alimentary Tract
Isolation and sequencing of a novel tropomyosin isoform preferentially associated with colon cancer,☆☆,

https://doi.org/10.1053/gast.2002.34154Get rights and content

Abstract

Background & Aims: Nonmuscle human tropomyosin (hTM) isoforms have distinct functions and may play important roles in various disease processes. Methods: In an attempt to identify colon epithelial tropomyosin isoform, a complementary DNA library prepared from a human colon cancer cell line T84 was screened by an oligonucleotide probe complementary to messages of all known hTM isoforms. A novel clone called TC22 was obtained. The amino acid sequence of TC22 isoform is identical to isoform 5 (hTM5) apart from the C terminal domain, amino acids 222–247 coding the exon 9. Results: Northern blot analysis showed that TC22 message is expressed in transformed epithelial cell lines and tumor tissues but not in normal epithelial cells. We developed a monoclonal antibody specific to TC22 isoform (TC22-4). By Western blot and immunoperoxidase assays, we analyzed 105 colonic specimens (fresh frozen and formalin fixed) from 96 patients with colon polyps (hyperplastic) or adenomas with or without dysplasia and cancer. Twenty-one of 22 (95%) of colon cancer specimens showed the presence of TC22, compared with only 1 of the 17 normal colon specimens and none of the 13 hyperplastic polyps (P < 0.0001). As assayed by immunoperoxidase staining, TC22 expression progressively increased in benign adenomatous polyps (35%) and polyps with mild and severe dysplasia (57% and 100%, respectively). Conclusions: We cloned and sequenced a novel hTM isoform, TC22, which is strongly associated with colonic neoplasia and carcinoma. TC22 may provide a useful biomarker for surveillance of colon cancer.

GASTROENTEROLOGY 2002;123:152-162

Section snippets

Isolation and characterization of cDNA clones

A λZAPII phage library prepared from T84 human colon cancer cell poly(A)+ RNAs was purchased from Stratagene (La Jolla, CA). A 29-mer oligonucleotide probe common to all of known hTM messenger RNAs was used to screen this library as described previously.4 After plaque purification, 28 positive clones were obtained. Plasmids from positive plaques were isolated from their Uni-ZAP XR vector by in vivo excision with helper phage as described by the manufacturer. The cDNA inserts were polymerase

Identification of cDNA clones encoding tropomyosin isoforms from T84 colon cancer cells

Previously, we designed a common oligonucleotide probe (REN29) and showed that this REN29 probe was capable of recognizing messenger RNAs derived from all 4 hTM genes.4 Using this probe to extensively screen a cDNA library prepared from T84 cancer cells, 28 positive clones were obtained. After restriction enzyme analysis, PCR amplification with isoform-specific primers (Table 1), and nucleotide sequencing of the inserts, 15, 11, and 1 clones were classified into hTM5, hTM4, and hTM1,

Discussion

In the present study, we cloned a novel tropomyosin TC22 isoform from a colon cancer cell line T84. This isoform has 5'-untranslated sequence and most of the coding sequences (except the last coding exon, exon 9) identical to that in hTM5, suggesting that both isoforms are derived from the same human γ-TM gene. Furthermore, the divergent coding sequence and 3'-untranslated sequence of TC22 have high homology (96.1% and 77%, respectively) to NM-4 tropomyosin isoform from rat cochlea.

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    Address requests for reprints to: Kiron M. Das, M.D., Ph.D., UMDNJ–Robert Wood Johnson Medical School, 1 Robert Wood Johnson Place, New Brunswick, New Jersey 08903. Fax: (732) 235-7792.

    ☆☆

    Supported by grants (NIDDK47673 and HD18577) from the National Institutes of Health. J.-R.Y. was partly supported by grant 2000-1-20200-003-1 from the Basic Research program of the Korean Science and Engineering Foundation. X.G. is supported in part by a research grant from the Crohn's and Colitis Center of New Jersey.

    J.L-C.L. and X.G. contributed equally to this work.

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