Identification of Genes W ith Specific Expression
in Pancreatic Cancer by cDNA Representational
Thomas M. Gress,1Christine W allrapp,1Marcus Frohme,3Friederike Mu ¨ller-Pillasch,1Ulrike Lacher,1
Helmut Friess,2Markus Bu ¨chler,2Guido Adler,1and Jo ¨rg D. Hoheisel3*
cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic
enrichment procedure for the isolationofdifferentially expressedgenes. Inthisstudy, the technique wasusedto isolate novel
genesspecificallyexpressedinpancreaticcancer.cDNA-RDA wasdoneoncDNA reversetranscribedfromapoly(A)1mRNA
pool madefrom10cancer tissues(tester) byusingasadriver acDNA fromapoly(A)1mRNA pool madefromacombination
of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy
pancreas mRNA inthe driver preparationeliminated the influence ofstromal tissue components present as contaminationin
the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These
were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed
homologiesto fivegenespreviouslyimplicatedinthecarcinogenesisofthepancreasor other tissues.Elevenfragmentshadno
significant homologyto anyknowngeneandthusrepresent novel candidatediseasegenes.Theexperimentsdemonstratethat
cDNA-RDA isareproducible andhighly efficient methodfor the identificationofnovel geneswithcancer-specific expression.
r1997 Wiley-Liss, Inc.
Pancreatic canceristhefifthmost commoncause
of cancer-related deathsin industrialized countries,
with a dismal prognosis, an increasing incidence,
and no or only ineffective means of treatment
(Myerset al., 1989;Murret al., 1994).T hedevelop-
ment of new treatment modalities and diagnostic
and preventive approachesrequiresan understand-
ing of the molecular mechanisms of the complex
multistep processof tumorigenesisin the pancreas.
Despite its importance as a health problem with a
major socioeconomic impact, the mechanisms of
carcinogenesis in pancreas are poorly understood.
T hus, it is of major importance to identify novel
genesinvolved in pancreatic carcinogenesis. In this
context, the analysis of differences between tumor
and healthy tissue at the level of mRNA offers a
very good prospect for the isolation of candidate
Two methods based on polymerase chain reac-
tion(PCR) areavailabletoidentify changesof gene
expression between cancer and normal tissues.
Differential-display PCR (DD-PCR; L iang and
Pardee, 1992) relies on random-primed amplifica-
tion of total RNA of two different populations,
tial bands in sequencing gels. Representational
difference analysis (RDA) is a combination of
subtraction and kinetic enrichment coupled to
subsequent amplification, originally developed for
genomic DNA toisolate differences between com-
plex genomes(L isitsyn et al., 1993, 1995). Whereas
DD-PCR amplifies fragments from all represented
mRNA species,RDA hastheadvantageofeliminat-
ing fragments present in both populations, leaving
only thedifferences. Recently, RDA protocolswere
adapted to the examination of differential gene
expression between two mRNA populations (Hu-
bank and Schatz, 1994). T his technique offers the
means to reduce rapidly the number of candidate
genes in a highly specific manner. T he aim of the
present study was to use RDA on cDNA popula-
tionstoisolate novel differentially expressed genes
of potential importance to biomedical analyses of
Supported by the German Ministry of Science and Education;
Contract Grant numberKBF 01GB 9401.
*Correspondence to: Dr. Jo ¨rg Hoheisel, Molecular-Genetic Ge-
nome Analysis, Deutsches Krebsforschungszentrum, Im Neuenhei-
mer Feld 506, D-69120 Heidelberg, Germany; e-mail: j.hoheisel@
Received 4September1996; Accepted 13November1996
GENES, CHROMOSOMES & CANCER 19:97–103 (1997)
r1997 W iley-Liss, Inc.
with other methods such as differential hybridiza-
tions (e.g., Gress et al., 1992). cDNA-RDA studies
the entire set of transcribed genes in one experi-
ment, whereas only much simpler subsets are ana-
lyzed simultaneously by the differential-display
technique(L iang andPardee, 1992). By including a
ratios, cDNA-RDA allowed not only the detection
of absolute differences in gene expression but also
variations in the scale of expression, which are the
most frequent events leading to cancer-specific
differential gene expression. T he number of genes
with altered expression in tumors should be more
than the 16 that have actually been identified. In
theDP2andespecially theDP1amplicons(Fig. 1),
indicating that this low number resulted from the
DP3. An influence of variable amplification rates
tion of poorly amplified DNA, cannot be ruled out
entirely, although the selectivity of the RDA pro-
cess should more than compensate for such an
effect. Besides the ability to detect differences in
expression levels, cDNA-RDA also seems well
suited to detect cancer-specific splice variants. In
summary, this study has shown the feasibility of
using cDNA-RDA in approaches aiming at the
isolationof cancer-specific expressedgenesandhas
led to the identification of a number of genes
senting novel candidatediseasegenes.
We thank K. Monn for valuable technical assis-
tance, D.G. Schatz (Yale University School of
Medicine, New Haven, CT ) for providing a de-
tailed protocol for cDNA-RDA, and H. Delius
many) for his advice on sequence analysis and
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DIFFERENTIAL EXPRESSION IN PANCREATIC CANCER