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MUC2
Gene Suppression in Human Colorectal Carcinomas and Their Metastases: In
Vitro Evidence of the Modulatory Role of DNA Methylation |
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Christoph Hanski,
Eva Riede, Alexei Gratchev, Hans Dieter Foss, Christian BAdohm, Enno Klu\Smann,
Michael Hummel, Benno Mann, Heinz Johannes Buhr, Harald Stein, Young S.
Kim, Jim Gum, and Ernst Otto Riecken |
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Abteilung
Gastroenterologie (CH, AG, CB, EK, EOR), Institut fAdur Pathologie (HDF,
MH, HS), and Abteilung Chirurgie (ER, BM, HJB) in UniversitAdatsklinikum
Benjamin Franklin der Freien UniversitAdat Berlin, Berlin, Germany; and
University of California (YSK, JG), Veterans Administration Center, San
Francisco, California |
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The development of the majority
of colorectal carcinomas is associated with a diminished expression of the
intestinal mucin MUC2 in the tumor cells. The significance and the mechanism
of this alteration are not yet known. We sought to determine the molecular
basis of this tumor-associated change and to investigate the extent to which
it might also relate to metastases. MUC2 gene expression was compared in
normal (N), carcinomatous (T), and metastatic tissues (M) from nine patients
by immunohistochemistry, in situ hybridization, and Northern blotting. Immunohistochemistry
and in situ hybridization showed consistently lower amounts of the expressed
protein and mRNA in T and in M than in N; quantitative analysis by Northern
blotting confirmed that the differences between MUC2 mRNA expression between
N, T, and M were significant, the expression in metastases being less than
5% of that in the normal colonic tissue. The influence of DNA methylation
as a possible regulatory mechanism of MUC2 gene expression was tested after
the 5` and 3`- regions flanking the first exon of MUC2 were recovered from
a genomic DNA library and used as probes in Southern blot. The DNA was isolated
from colon carcinoma cell lines expressing MUC2 strongly (LS174T) or moderately
(T84) and from that which was nonexpressing (Colo 205), and it was digested
with the methylation-sensitive enzyme HpaII. The Southern blot patterns
indicated that the increased methylation in the promoter region was concomitant
with the decrease of MUC2 mRNA expression. Methylation of the promoter region
ligated into a reporter vector suppressed the expression of the luciferase
reporter gene in the three investigated cell lines. Furthermore, the expression
of MUC2 gene was enhanced by treating the MUC2-expressing colon carcinoma
cells with 5-aza-2`-deoxycytidine, a methylation-inhibiting agent. To our
knowledge this is the first report to show that: (a) MUC2 gene is strongly
suppressed in liver and lymph node metastases of colorectal carcinomas,
and (b) suppression of MUC2 gene in colon carcinoma cells in vitro is associated
with methylation of the promoter region. |
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