PCR EXPRESSION ANALYSIS OF THE ESTROGEN-INDUCIBLE GENE BCEI IN GASTROINTESTINAL AND OTHER HUMAN TUMORS

A pol ymerase chain reaction (PCR) assay was deve loped to test for tumor cell speci fic expression of the BCEI gene, This new marker gene. reported at first for human breast cancer. was found spec ificall y active in various gastrointestinal carcinomas by pre viousl y applying immunohistochemistry and RNA (Northern blot) analysis. Presently . by using re verse tran scription -PCR analysis. a series of primary tumor ti ssues and established tumor cell lines were testcd for BCEI transcription. This approach was compared to illlmunostaining achi eved by an antibody directed against the BCEI genc's product. The result demonstrate the superior sensitivity of PCR by indicati ng the gene' s expression in cases where i mmunohistochemicaltcsti ng remained negati vc.


INTRODUCTION
In many regulatory proteins, cysteine-rich regions responsible for protein-protein interaction arc present.Within the last few years a new cysteine-rich structure.known as either the trefoil moti I' or P-domain , has been described (Thim 1989).The P-domains are ca.50 amino acid residues in length; the 6 cysteine residues form 3 intramolecular disul fide bridges and hence 3 loops which are responsibl e for the structure' s resistance against proteolytic degradation.P-domains werc discovered in frog mucins and porcine.murine and human peptides from the gastrointestinal tract (Hoffmann & Hauser 1993).All known P-domain-containing peptides display very distinctive expression patterns in tissues of normal individuals.Their precise physiological role however is not yet fully understood.
For convenient screening of large sample collections and in cases of limited sample size we now have developed a reverse transcription-polymerase chain reaction (RT-PCR) assay and applied this method to monitor the BCEI gene's activity in tumors and permanent cancer cell lines from the gastrointestinal tract.Such a strategy has been already successfully applied for various gene expression studies such as for leukemia specific BCR-ABL fusion mRNA and Duchenne muscular dystrophy mutations (Kawasaki (!f al., 1988, Roberts (!f (II., 1990).

RT-PCR
Total cellular RNA was extracted by a standard procedure (Sambrook et al 1989).The reverse transcription-PCR steps wcre described previously (Roberts (!f (II. , 1990) and were applied with the following modification.

RESULTS
The BCEI (pS2) gene, originally shown to be under estrogen transcriptional control and isolated from the breast cancer cell line MCF7, displays within its coding DNA sequence blocks suitable to generate primers necessary for PCR.From two, primers were synthesized and the complete RT-PCR assay was performed with mRNA from MCF7 cells.
An expected product of 150 bp was obtained with as little as 100 ng total RNA (corresponding to ca. 5 ng mRNA) ascertaining the sensitivity of this approach (Fig. 1).In addition to MCF7 cells, containing estrogen receptors (leading to the beforementioned transcriptional control of pS2), we also tested an estrogen receptor-negative breast cancer cell line, MDA/MB23I.A distinct 150 bp band in the RT -PCR assay demonstrated pS2 expression in these cells lacking estrogen receptors (Fig. I).
Our previous studies (by immunostaining and Northern blot hybridization) revealed that the BCEI gene was transcriptionally inactive in normal pancreatic cells and we noted its expression in a primary pancreatic cell culture plus in a majority (74 %) of pancreatic tumor samples.Weaker but still distinguishible immunostaining «5 % immunopositive cells) was notable in the remaining 26% of the tumors.We decided to investigate a series of permanent cell lines established from pancreatic carcinoma.Surprisingly, none of them showed BCEI express ion when tested by immunostaining.PCR analysis resulted in clearly visible signals in 3 out of 5 investigated cell lines (Table I).We then extended the PCR study to additional carcinoma cell lines from colon and endometrium and a primary culture from a pancreatic carcinoma, the latter noted to display distinct immunostaining (Fig. 2).The analysis confirmed pS2 expression in the primary culture whereas the endometrium and colon carcinoma cell lines remained negative.
In stomach carcinoma of the intestinal type , previously described to often display none or weak BCEl expression, antibody-staining indicated -30% immuno-positive cells in one sample and remained very weak or negative in 4 out of 5 cases.However, residual gene expression must be present below the immunodetection level in all the cases since RT-PCR analysis showed bands of correct size (Table I) .Following the milestone in genomic analysis, the application of PCR to define DNA landmarks on the physical map of the human genome (STS's), as advocated by Olson et aI., (Olson et aI., 1989), the PCR approach was extended to include RNA as a starting template.Synthesis of cDNA from a mRNA pool followed by PCR using selected primers allowed detection of specific fusion gene products in human leukemia (Kawasaki et al.. 1988).Subsequently, this assay for the Philadelphia chromosome was shown to be very sensitive (down to mere 5 affected cells) (Morgan et al.. 1989).To investigate the function and regulation of the BCEI gene we established a similar assay to conveniently screen for cell-type specific transcription.Using two apt primers, BCEI-mRNA was easily detectable in MCF7 cells, a cell line originally used for isolation of the BCEI gene.Since it is specifically expressed in various gastrointestinal tumors, its activity was presently monitored in these cells by PCR and immunostaining.To our surprise, some of the permanent cell lines established from colon, endometrium and pancreatic carcinoma showed no BCEI transcription.In 3 pancreatic cell lines, histochemical detection was not sufficiently sensitive but, by PCR, we were able to prove the gene ' s transcription.The same seems to be true for the gastric carcinoma samples of the intestinal type: PCR appeared superior in detecting the pS2 gene's transcription at lowest levels.
On the basis of data accumulated from studies performed by us and other groups (reviewed in Hoffmann & Hauser 1993) the evidence points towards BCEl's (and some other P-domain peptides') role in vivo as either a growth modulator or as a control molecule in maintaining surface integrity of mucous epithelia.Immortalized cancer cell lines obviously can loose the ability to express this gene or, at least, reduce the gene's activity below levels of immunodetection.An apt PCR assay, as demonstrated , will allow a more sensitive differentiation between complete shut-off, residual expression, and gene induction.Such determination appears essential for studies of BCEI's function , in particular in transgenic cells.
TableI.Expression of BCEI in primary tumors and permanent cell lines as determined = estrogen receptor positive cells ER-= estrogen receptor negative cells +1-= very weak immunostaining (in <5% of the cells)

Figure 2 :
Figure 2: Immunodctcction of the BeEI protein expressed in pancreatic carcinoma cells maintained as a primary cell culturc.