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Retinoic acid receptor beta regulates growth and differentiation in human pancreatic carcinoma cells
GASTROENTEROLOGY 1997;113:920–929
Retinoic Acid Receptor b Regulates Growth and Differentiation in Human Pancreatic Carcinoma Cells ASTRID KAISER,* HERMANN HERBST,‡ GARY FISHER,§ MICHAEL KOENIGSMANN,\ WOLFGANG E. BERDEL,‡ ERNST–OTTO RIECKEN,* and STEFAN ROSEWICZ* Departments of *Gastroenterology, ‡Pathology and \Hematology/Oncology, Klinikum Benjamin Franklin, Berlin, Germany; and §Department of Dermatology, University of Michigan, Ann Arbor, Michigan
Background & Aims: Retinoic acid receptor b (RARb) expression is lost or decreased during malignant transformation in human pancreatic adenocarcinoma. The aim of this study was to evaluate the role of RARb expression in the propagation of a malignant phenotype in human pancreatic carcinoma cells. Methods: Overexpression of RARb in the human pancreatic carcinoma cell line DAN-G was achieved by selecting stable transfected cell clones. Genomic integration and expression were verified by Southern and Northern blotting and electrophoretic mobility shift assays. Growth was determined by cell number and xenografts transplanted into nude mice. Differentiation was examined by immunohistochemistry. Results: Overexpression of RARb in DAN-G cells inhibited cellular proliferation in vitro and in vivo. Furthermore, RARb overexpression resulted in induction of cellular differentiation in xenografted tumors as evidenced by increased tumor cell expression of duct cell differentiation markers carcinoembryonic antigen (CEA), CA19-9, and cytokeratin 7. Conclusions: Decreased expression of RARb plays a key role in the maintenance of a malignant phenotype in human pancreatic adenocarcinoma and therefore represents a novel target for experimental strategies in the treatment of pancreatic cancer patients.
A
denocarcinoma of the exocrine pancreas accounts for 25% of all gastrointestinal tumors and currently ranks as the fifth most common cause of cancer-related deaths in Western countries.1 Increasing incidence and lack of effective treatment options have resulted in a widespread therapeutic nihilism. Operative resection represents the only curative approach; however, at the time of diagnosis more than 90% of pancreatic cancer patients present with advanced regional disease or distant metastasis and can therefore not be considered for surgery.2 Conventional chemotherapy has not resulted in a significant survival benefit over the last 30 years, and the average survival time is still measured in months with a 5year survival rate of õ 2%.3 Based on these observations, it becomes clear that only a more detailed understanding of the tumor biology of pancreatic cancer will reveal novel / 5e20$$0042
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cellular targets for experimental therapeutic strategies. In this context, we have previously observed that natural and synthetic derivatives of vitamin A, biochemically summarized as retinoids, might offer a therapeutic alternative in pancreatic cancer patients. This is based on experimental evidence showing that retinoid treatment of human pancreatic carcinoma cells in vitro and in vivo results in a profound inhibition of tumor cell proliferation and induction of tumor cell differentiation.4 Retinoids exert these intriguing effects through interaction with specific nuclear receptors. Based on molecular cloning studies to date, two families of nuclear retinoid receptors have been described, each consisting of three receptor subtypes, a, b, and g: the retinoic acid receptors (RAR) which bind the naturally occurring retinoid all-trans retinoic acid (ATRA) with high affinity5,6; and the retinoid X receptors (RXR) whose naturally occurring, biologically active ligand is 9-cis retinoic acid, a geometric isomer of ATRA.7 The ligand-binding domains between these two retinoid receptor families share only a 29% sequence homology.8 In addition, each RAR/RXR gene generates multiple isoforms by either differential use of internal promotors or alternative splicing of exons.9 Both receptor families act as ligand dependent transcription factors, controlling gene transcription initiated from promotors of retinoid regulated genes by interacting with cis-acting DNA elements, the so-called retinoic acid responsive elements (RAREs).9,10 This multiplicity of receptors and gene pathways explains the diverse effects of retinoids on a wide range of cellular processes. Tissue-specific restricted expression of RAR/RXR subtypes and isoforms during embryogenesis and in the adult organism suggests that each RAR and RXR subtype exerts a unique biological function.11 Furthermore, Abbreviations used in this paper: ATRA, all-trans retinoic acid; bp, base pair; CEA, carcinoembryonic antigen; Ck, cytokeratin; CMV, cytomegalovirus; FCS, fetal calf serum; RAR, retinoic acid receptor; RARE, retinoic acid responsive elements; RT-PCR, reverse-transcription polymerase chain reaction; RXR, retinoid X receptors. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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disruption of the intracellular retinoid pathway via interference with specific RAR subtypes can result in carcinogenesis.12 – 14 Summarized with our observation that retinoids exert antiproliferative effects and induce differentiation in human pancreatic carcinoma cells4 we hypothesized that a deregulation in RAR subtype expression might be involved in the propagation of the malignant phenotype of human pancreatic cancer. In a subsequent in situ hybridization study, we analyzed RAR subtype expression in human pancreatic carcinoma tissue of 24 patients.15 Although there was no difference in the expression of RAR a and g between normal and malignant tissue, we observed that approximately one third of all pancreatic tumors completely lost the expression of RARb when compared with their nontransformed counterparts. Furthermore, the remaining tumors expressed significantly less RARb messenger RNA (mRNA) transcripts than adjacent normal pancreatic ductal cells. Moreover, we observed a tight correlation between the loss of RARb expression and the degree of cellular dedifferentiation.15 These data suggested that loss or decreased expression of RARb could either be an innocent epiphenomenon associated with malignant transformation or might indeed play a central role in the propagation of the malignant phenotype in human pancreatic adenocarcinoma. To dissect this problem we chose a reverse approach by overexpressing RARb in the human pancreatic tumor cell line DAN-G. We now show that restoration of RARb expression in human pancreatic tumor cells results in growth inhibition and induction of cellular differentiation.
Materials and Methods Materials The human pancreatic carcinoma cell line DAN-G was obtained from Deutsche Krebsforschungszentrum (Heidelberg, Germany). RPMI medium was obtained from GIBCO (Berlin, Germany), and charcoal-stripped, heat-inactivated fetal calf serum (FCS) from Biochrom (Berlin, Germany). All retinoids were provided by Hoffmann-LaRoche (Basel, Switzerland). We obtained [a-32P]-deoxycytidine triphosphate (6000 Ci/mmol) from DuPont (Bad Homburg, Germany). Random priming labeling kit and HybondN/ were obtained from Amersham (Braunschweig, Germany); nitrocellulose and blotting paper were purchased from Schleicher & Schuell (Dassel, Germany); Poly AT Tract isolation kit was obtained from Serva (Heidelberg, Germany); RNA and DNA molecular size markers, G418, and proteinase K were obtained from Bethesda Research Laboratories (BRL, Bethesda, MD); restriction enzymes from Boehringer Mannheim (Mannheim, Germany). The pRC-CMV mammalian expression vector was obtained from Invitrogen (La Jolla, CA). All other chemicals were of analytical grade and purchased from Sigma (Deisenhofen, Germany).
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Cell Culture and Growth Assays DAN-G cells were grown as subconfluent monolayer cultures supplemented with 10% (vol/vol) charcoal-stripped, heat-inactivated FCS penicillin (100 U/mL), and streptomycin (100 mg/mL). The cells were maintained under 95% air and 5% CO2 at 377C. All experiments were performed in the log phase of growth after the cells had been plated for 24 hours unless otherwise stated. Cell viability was routinely checked by trypan blue exclusion test and was consistently found to be ú95%. After mild trypsinization, cells were plated in 22 mm culture dishes at a density of 50,000 cells/well in the presence of RPMI containing 10% FCS. At the indicated times, cells were washed with 154 mmol/L NaCl and then obtained by trypsinization. Cells were then resuspended in phosphate-buffered saline to ensure a single cell suspension. Viable cells were counted in a hemocytometer by trypan blue exclusion. Triplicate wells were analyzed for each time point.
Stable Transfection of DAN-G Cells The 1.5-kilobase (kb) complementary DNA (cDNA) of human RAR-b2 (formerly described as RAR-b06) was isolated from the host vector pSG5 using SacI and BamHI restriction digests and subcloned in the linearized HindIII site of pRC/ CMV vector, containing a neomycin resistance gene. This yielded the RAR-b2 cDNA preceded by the cytomegalovirus (CMV) promotor and followed by the human growth hormone termination and polyadenylation signal. The construct was confirmed by restriction analysis. DAN-G cells, (approximately 4 1 106 cells per 100 mm dish) were transfected with 5 mg of pRC-b2 plasmid using the Lipofectamine Reagent (GIBCO BRL, Bethesda, MD) following the instructions supplied by the manufacturer. Control cells were transfected with vector alone (mock-transfected) not containing RARb-specific nucleotide sequences. Twenty-four hours after transfection, cells were diluted 1:10 and plated in medium containing 1 mg/mL G418. Resistant cell clones appeared after approximately 21 days and were picked for expansion at 35 days. The RARb and mock-transfected cell clones were maintained under selection pressure for all the experiments.
Southern Blotting For isolation of genomic DNA from transfected and parental cells, 3–4 1 107 cells were collected by trypsinization and centrifugation. The pellet was resuspended in 100 mmol/ L NaCl, 10 mmol/L Tris-HCI pH 8, 25 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate, and 1 mg/mL proteinase K and incubated with shaking at 507C for 12 hours. Samples were extracted twice with an equal volume of phenol/chloroform/isoamylalcohol (vol/vol 25:24:1) and treated with ribonuclease A (10 mg/mL) at 377C for 4 hours. DNA was precipitated with 1/10 volume sodium acetate and ethanol at room temperature. After low-speed centrifugation the pellet was rinsed with 70% ethanol. Fifty micrograms of genomic DNA were digested with EcoRI and separated on a 1% agarose gel at 47C overnight. After denaturation, DNA
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was transferred to HybondN/ and prehybridization and hybridization was performed exactly as previously described.4
RNA Isolation and Northern Blotting RNA was isolated by a modification of the method of Chirgwin et al16 exactly as previously described.4 Poly(A)/purified RNA was isolated using the Poly AtTract kit following the instructions supplied by the manufacturer. Qualitative analysis of RNA was performed using Northern blot analysis exactly as previously described.4 RNA was denatured by formaldehyde, subjected to electrophoresis through a 1% agarose gel in the presence of formaldehyde, and then transferred to nitrocellulose. An RNA ladder was electrophoresed in parallel for size determination. The RARb cDNA insert was electroeluted and then radioactively labeled using the random primer labeling kit following the instructions provided by the supplier. Unincorporated nucleotides were removed by passing the reaction sample over a Sephadex G 50 column (Sigma, Deisenhofen, Germany). The specific activity of the probes was routinely 1–2 1 109 cpm/ mg DNA. Filters were prehybridized and hybridized exactly as previously described.4 After hybridization, filters were sequentially washed with increasing stringency as previously described.4 Filters were then blotted dry and exposed to x-ray film for 2 days using two intensifying screens.
Electrophoretic Mobility Shift Assays Nuclear extracts from DAN-G cells were prepared by using a micropreparation technique. Five micrograms of nuclear protein extracts were incubated on ice for 15 minutes with 2 mg of poly[d(I-C)]/poly [(I-C)] in 20 mmol/L TrisHCL pH 7.9, 1 mmol/L EDTA, 0.1% NP-40, 1 mmol/L dithiotreitol, 6.25% Ficoll, and 0.1 mg/mL bovine serum albumin (BSA) in a final reaction volume of 25 mL. A doublestranded oligideoxynucleotide (5*-TCGACTAAGGGTTCACCGAAAGTTCACTCGCA-3*) containing the retinoic acid response element in the RARb2 promotor was used as a probe. The oligonucleotide was end-labeled with [g-32P]ATP by a T4 polynucleotide kinase reaction, and unincorporated nucleotides were removed by passing the reaction mixture over a DE52 anion exchange column (Whatman, Maidstone, England). We added 0.1 ng radioactively labeled oligonucleotide to each reaction and incubated for 30 minutes at room temperature. For competition experiments a 40-fold excess of unlabeled oligonucleotide was added to the reaction mixture before the addition of labeled oligonucleotide. The samples were electrophoresed on a 6% polyacrylamide gel. Radioactive proteinDNA complexes in the gels were quantitated using a PhosphorImager (Berthold, Berlin, Germany).
Tumor Growth in Nude Mice Four-week-old female athymic BALB/c nu/nu mice were obtained from Bomholtgard Ltd. (Ry, Denmark). Mice were maintained under specific pathogen–free conditions and fed an autoclaved standard diet (Altromin 1324; Altromin, Lage, Germany) and sterilized water at low pH. Human pancreatic tumor cells were xenotransplanted intracutaneously into
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the flank as single cell suspensions in 0.1 mL phosphate-buffered saline. Equal amounts (5 1 105) of viable wild-type and RARb transfected cells were injected into each animal. Tumor growth was measured with calipers at days as indicated. Mean diameter (length plus width/2) is given as tumor size in millimeters.
Histological Analysis of Xenografts Tumors were immediately removed after killing the animals. Four-micrometer serial sections of formol-fixed/paraffin-embedded tissue blocks mounted on 3-aminopropylethoxysilane-treated slides were stained by the immunoalkaline phosphatase method.17 Affinity-purified rabbit anti-mouse immunoglobulin antibodies and immunoalkaline phosphatase complex (diluted 1:20) were obtained from DAKO (Glostrup, Denmark). Throughout these studies, controls consisted of substitution of the primary antibody by normal serum. The following primary antibodies were used for immunohistochemistry: monoclonal antibody against carcinoembryonic antigen (CEA)18 obtained from DAKO (Glostrup, Denmark) and monoclonal antibody against cytokeratin 719 obtained from Boehringer Mannheim (Mannheim, Germany). Monoclonal antibodies specific for the carbohydrate antigen CA19-920 and for the proliferation-associated antigen Ki-67 (clone mib-1)21 were from CIS Diagnostik (Dreieich, Germany) and Dianova (Hamburg, Germany), respectively.
Statistics Results were evaluated by repeated measurement twoway analysis of variance with a multiple contrast test (Hotelling T2). P values of õ0.05 were considered to be significant.
Results Generation of a RARb Overexpressing DAN-G Cell Clone Integration of transfected RARb DNA into the genome after stable transfection was investigated by Southern blotting. As shown in Figure 1, probing the EcoRI-digested genomic DNA from the wild-type parental, mock-transfected and RARb transfected cells with a RARb cDNA, revealed an identical fragment hybridization pattern corresponding to the endogenous RARb gene in all cell lines. Only RARb transfected cells however showed additional bands of 1300 base pairs (bp), 610 bp, and 190 bp, which had to be expected from the internal EcoRI restriction fragment size of the transfected RARb cDNA. Thus, a DAN-G cell clone was established with stable integration of the RARb in its genome. We then used Northern blot analysis to investigate mRNA transcription from the integrated DNA. Hybridization with the RARb cDNA showed a strong signal of approximately 2.0 kilobases in the RARb transfected DAN-G cell line, which corresponds to the expected size of the transcribed mRNA for RARb2 WBS-Gastro
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(Figure 2). In contrast, no significant hybridization signal for RARb could be observed in the parental wildtype and mock-transfected DAN-G cells (Figure 2). This is in agreement with our previous observation that parental DAN-G cells express trace amounts of RARb mRNA, which can only be detected by the highly sensitive reverse-transcriptase polymerase chain reaction (RT-PCR) technique but can barely be detected by conventional Northern blotting using poly(A)/ purified RNA.4 Transfection of RARb cDNA had no effect on RARa and RARg mRNA expression when compared with mock-transfected controls or wild-type cells (data
not shown). Therefore, this DAN-G cell clone shows selective overexpression of RARb mRNA compared with the parental wild-type and mock-transfected controls. Of the initially selected 4 cell clones, 3 of 4 revealed genomic integration of RARb but only 1 of 4 clones showed appreciable RARb mRNA overexpression. This cell clone was used for all further studies. To verify that the transfected RARb mRNA is translated into a functionally active protein, we performed electrophoretic mobility shift assays using a RARE in the RARb2 promotor as a probe. As seen in Figure 3, RARb transfected DAN-G cells display a 4–5-fold stronger RARE binding as compared with wild-type or mock-transfected controls, because of overexpression of a functionally active RARb protein. Effects of RARb Overexpression on DAN-G Cells In Vitro We next investigated the effects of RARb transfection on cellular proliferation in DAN-G cells. Overexpression of RARb results in a significant decrease of tumor cell growth compared with mock-transfected or untransfected control cells (Figure 4). This inhibition
Figure 1. Genomic integration of RARb in transfected DAN-G cells. After stable transfection, EcoRI-digested DNA from wild-type, mocktransfected, and RARb transfected DAN-G cells was analyzed by Southern blotting using the radioactively labeled cDNA probe for RARb2 . The expected fragment sizes are deduced from the internal EcoRI sites (E) of the transfected RARb nucleotide sequence (190 and 610 bp). The 1300-bp fragment corresponds to the next EcoRI restriction site in the DAN-G cell genome. A shorter exposition of the autoradiograph revealed a 1200-bp fragment size corresponding to the endogenous and a 1300-bp fragment size corresponding to the transfected RARb. A longer exposition of the autoradiograph had to be chosen to visualize the weaker 190-bp fragment. Molecular size determination was obtained from a DNA ladder that was electrophoresed in parallel. The somewhat weaker hybridization signal in mocktransfected DAN-G cells is because of a lesser amount of digested DNA as evidenced from the ethidium bromide stain of the gel. Shown is a representative of two independent experiments.
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Figure 2. Expression of RARb mRNA in transfected DAN-G cells. Twenty micrograms of poly(A)/ purified RNA from wild-type, mocktransfected, and RARb transfected DAN-G cells were separated electrophoretically and then analyzed by Northern blotting using the random primed cDNA probe for RARb. Molecular size was determined by an RNA ladder, electrophoresed in parallel. Only RARb transfected DAN-G cells expressed a clearly visible 2.0-kb mRNA corresponding to RARb. Shown is a representative of two independent expriments, yielding identical results.
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Effects of RARb Overexpression on DAN-G Cells In Vivo
Figure 3. Electrophoretic mobility shift assay of RARb transfected DAN-G cells. EMSAs using a double-stranded oligonucleotide containing the RARE of the RARb2 promotor were performed in the presence (/) or absence (0) of a 40-fold excess of cold oligonucleotide. RARb overexpressing DAN-G cells display an increased binding of nuclear extract proteins to the RARE oligonucleotide compared with mock-transfected and wild-type controls. Shown is a representative of three independent experiments, yielding almost identical results.
of growth in the absence of endogenous (charcoalstripped, heat-inactivated FCS) and exogenous retinoids could be observed as early as 48 hours and remained significant for up to the investigated time period of 5 days. In contrast, transfection of DAN-G cells with plasmid alone, devoid of RARb nucleotide sequence (mock-transfected), did not result in growth inhibition compared with the parental wild-type, indicating that overexpression of RARb is specifically responsible for the observed growth retardation. Furthermore, overexpression of RARb had no significant effect on the growth inhibition mediated by ATRA when compared with mock-transfected or wild-type DANG cells (cell numbers after 120 hours of 10 mmol/L ATRA were 62% { 5% [n Å 5] and 67% { 8% [n Å 6] of untreated controls for mock-transfected and RARb transfected, respectively). / 5e20$$0042
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Subsequently, we have evaluated putative tumor growth retardation by RARb overexpression in xenografts of DAN-G cells in nu/nu BALB/c mice in vivo. Preliminary experiments showed a reliable and reproducible tumor formation using this cell line at a cell load of 5 1 105 cells per mouse. For these experiments we compared wild-type and RARb transfected cells, because in the previous experiments no difference in terms of growth characteristics and morphological changes were observed between mock-transfected and wild-type DANG cells. We observed some remarkable differences between the two experimental groups. For one, nude mice injected with wild-type DAN-G cells showed a rather rapidly developing cachexia as compared with the animals bearing RARb transfected DAN-G tumors. This observation was reflected by whole animal weight determined 15 days after tumor cell injection, which was 15.61 { 3.15 g (n Å 10) in wild-type injected versus 18.71 { 1.21 g (n Å 11) in animals that had been injected with RARb overexpressing DAN-G cells. However, these differences did not reach statistical significance. In accordance with our in vitro data, we observed a significant growth retardation of RARb transfected DAN-G cell xenograft tumors compared with the untransfected controls (Figure 5), the differences becoming statistically significant at day 13 after the injection. Effects of RARb Overexpression on Tumor Cell Proliferation and Differentiation In Vivo Histological analysis of the tumors showed the appearence of a dedifferentiated adenocarcinoma with a
Figure 4. Effects of RARb overexpression on DAN-G cell proliferation in vitro. DAN-G cells (mock-transfected and RARb transfected) were plated at a density of 50,000 cells per well in triplicates. Viable cell number was determined by counting cells after trypan blue exclusion at the indicated time periods. Cell viability ú 95% was required before the experiment was evaluated. The growth curve of mock-transfected and parental DAN-G cells was identical. Shown is the MEAN { SEM of five independent experiments, each performed in triplicate (*P õ 0.05).
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the mucin carbohydrate epitope Ca19-9 showed a moderately strong and diffuse staining pattern in control tumors (Figure 7C). However, in RARb overexpressing xenografts a very strong Ca19-9 expression could be detected in the nonproliferating, CEA-expressing cystic tumor areas (Figure 7D). The intermediate filament protein cytokeratin 7 (Ck7) was only expressed sporadically in very few single cells of nontransfected control tumors (Figure 7E), whereas we observed significantly increased expression in the areas of presumed differentiation (Figure 8F). Furthermore, we observed a restricted overexpression of another ductal epithelial differentiation marker, the mucin MUC-1 in the nonproliferating cystic areas of RARb transfected xenografts, basically following the expression pattern of CEA (data not shown). Control
Figure 5. Effects of RARb overexpression on tumor growth in vivo. Xenograft tumor growth of wild-type and RARb transfected DAN-G cells was monitored at the indicated time points as outlined in Materials and Methods. Shown are the MEAN { SEM of 10 (wild-type DAN-G cells) and 11 (RARb transfected DAN-G cells) animals (*P õ 0.05).
modest stromal reaction (Figure 6). No major infiltration with hematopoietic or lymphocytic murine cells was observed in both groups (Figure 6). In comparison to the control tumors, we observed multiple cystic structures containing aggregates of tumor cells in the RARb transfected xenografts (Figure 6C and D). We then determined the fraction of actively proliferating tumor cells by immunohistochemistry using the mib-1 antibody directed against the proliferation-associated antigen Ki67.21 In control tumors, more than 90% of all tumor cells stained positively for mib-1, indicating a highly aggressive tumor biology (Figure 6E); in contrast, the fraction of proliferating cells in RARb transfected xenografts was remarkably reduced to approximately 50%, showing virtually no staining in the cystic structures, which were exclusively noted in the RARb transfected xenografts (Figure 6F). To investigate whether the inhibition of growth in RARb transfected xenografts was paralleled by induction of cellular differentiation, we performed immunohistochemistry with a panel of antibodies directed against molecules, commonly associated with increased duct cell differentiation. CEA was expressed at barely detectable concentrations in control tumors (Figure 7A). However, in RARb transfected xenografts, we observed a very strong CEA expression, which was focused and mainly restricted to the cystic nonproliferating areas, shown to stain negative for mib-1 (Figure 7B). Antibodies against / 5e20$$0042
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Figure 6. Effects of RARb overexpression on tumor morphology. Histological analyses and immunohistochemistry of (A, C, and E ) control tumors and (B, D, and F ) RARb overexpressing xenografts were performed as outlined in Materials and Methods. (A–D) H&E staining of tumor sections revealed the appearence of multiple cystic areas with tumor cell aggregrates (arrows in B and F ), which were only observed in RARb overexpressing xenografts. (A and C ) Control tumors displayed a solid dedifferentiated adenocarcinoma. Immunohistochemistry using the monoclonal antibody mib-1, directed against the proliferation associated Ki-67 antigen, revealed almost homogenous tumor cell staining in control tumors (E ), whereas the cystic tumor areas in RARb overexpressing xenografts were predominantly negative (delineated by arrows in F ). Shown is a representative analysis of 4 animals for each experimental group, which all revealed similar results (original magnification: A and B, 661; C and D, 1321; E and F, 661).
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Figure 7. Effects of RARb overexpression on tumor differentiation. Immunohistochemistry using monoclonal antibodies against (A and B ) CEA, (C and D ) CA 19-9, (E and F ) and cytokeratin 7 (CK 7) of (A, C, and E ) control tumors and (B, D, and F ) RARb overexpressing xenografts was performed as outlined in Materials and Methods. Shown is the pronounced increase in the expression of CEA, CA 199 and Ck 7 in the cystic, nonproliferating tumor areas of RARb overexpressing xenografts (arrows in B, D, and F ). Ck 7 stained only very few single cells in control tumors (arrow in E ). Shown is a representative of four animals for each experimental group, all yielding very similar results. (Original magnification: A-B, 661; C-D, 131; E-F, 661.)
antibodies irrelevant for pancreatic duct cell differentiation (Ck 20 and synaptophysin) stained consistently negative in control and RARb transfected tumors, confirming the specificity of the observed expression for the differentiation associated markers. Taken together, these immunohistochemical data suggest that overexpression of RARb in DAN-G xenografts results in multiple focal areas of tumor growth inhibition and induction of cellular differentiation.
Discussion We have previously shown that retinoids inhibit tumor cell proliferation and induce cellular differentiation in human pancreatic carcinoma cells in vitro and in vivo.4 In addition, we observed that RARb expression is either lost or significantly decreased in human pancreatic carcinomas when compared with the nontransformed human pancreas.15 This observation is reflected by in vitro / 5e20$$0042
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experiments, showing a significantly higher RARb expression in the well-differentiated human pancreatic tumor cell line Capan 122 (grade I according to the grading system by Kern et al.23) compared with the poorly differentiated tumor cell line DAN-G (grade III), in which RARb is only expressed at very low levels and exclusively detectable by the highly sensitive RT-PCR technique.4 This tight correlation between cellular differentiation and RARb expression in vivo and in vitro suggested that RARb might play an important role in the propagation of the malignant phenotype in human pancreatic adenocarcinoma. To investigate this hypothesis, we conferred RARb overexpression to the poorly differentiated human pancreatic tumor cell line DAN-G and examined the consequences in respect to tumor biology of this cell line. The RARb gene consists of four isoforms, generated by differential splicing, as well as differential use of promotors.24 In humans, the RARb3 equivalent does not appear to exist and RARb1 expression is principally limited to fetal cells and small cell lung cancer cells.12 The RARb4 isoform has been shown to act as a tumor promoter in lung and breast tissue of transgenic mice25 and is not expressed in human pancreatic carcinoma cell lines.4 We therefore decided to overexpress the RARb2 isoform, which also is the predominant isoform expressed in human pancreatic carcinoma cells.4 Selection of DAN-G cells, stably transfected with the RARb2 cDNA under control of the CMV promotor, showed one cell clone with stable integration of RARb nucleotide sequence in its genome and considerable overexpression of RARb mRNA transcripts and protein as judged by Southern and Northern blot analysis as well as electrophoretic mobility shift assays. Overexpression of RARb resulted in growth inhibition of DAN-G cells in vitro. This antiproliferative effect could be observed in the absence of biologically relevant retinoid concentrations (heat-inactivated, charcoalstripped FCS contains õ10013 mol/L ATRA). This observation confirms recent studies, suggesting that ligand occupancy is not always necessarily required for retinoid receptor-mediated transcriptional activation12,26 and that, for example, phosphorylation alone (e.g., caused by the addition of serum) can activate retinoid-receptor mediated transcriptional regulation.27 This is further supported by RARb overexpression experiments in human epidermoid lung cancer cells, showing that RARb overexpression alone, in the absence of retinoids, can induce antiproliferative effects.12 One possible explanation for this phenomenon is based on the observation that RAR can form a nonproductive complex with the proliferation associated AP-1 transcription factor, constituted by c-Jun homodimers or c-Jun/c-Fos heterodimWBS-Gastro
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ers.28,29 This experimental evidence provides a simple mechanism by which the intracellular concentration of RAR might limit cell growth and malignant progression. Whether phosphorylation or RARb/AP-1 interaction is indeed responsible for the observed growth inhibition in RARb overexpressing DAN-G cells is currently the subject of further investigation. When xenografted into nude mice, RARb overexpressing DAN-G tumor-bearing animals did not suffer to the same extent from the pronounced weight loss and cachexia observed in the control animals. We currently do not understand the molecular basis for this observation. However, it seems conceivable that RARb overexpression, which results in inhibition of growth and induction of cellular differentiation in vivo, mediates decreased expression of systemic acting factor(s) synthesized in the tumor cell, which might be responsible for the observed tumor-associated cachexia. In analogy to the in vitro experiments, we observed a significantly decreased growth rate of RARb overexpressing DAN-G cell tumors when xenografted in nude mice. Histological analysis showed multiple areas with a cystic appearence of tumor cell aggregates in RARb overexpressing tumors; immunohistochemistry, using an antibody against the proliferation-associated antigen Ki67, showed that the majority of these cells were not actively proliferating, compared with the control tumors where virtually more than 90% of all tumor cells stained positive for Ki-67. Given that inhibition of growth represents a hallmark of cellular differentiation, we investigated the differentiation state of these nonproliferating tumor cells using a panel of antibodies directed against antigens commonly associated with cellular differentiation of pancreatic carcinomas. The mucin carbohydrate epitope detected by monoclonal antibody Ca19-9, which is also expressed in normal ductal epithelium, was considerably stronger expressed in the nonproliferating tumor cell areas of RARb overexpressing xenografts. Expression of this antigen is commonly associated with well-differentiated pancreatic tumors and either only sporadically expressed or absent in poorly differentiated carcinomas.30,31 CEA, which was barely detectable in control tumors, was also very strongly expressed in the nonproliferating cystic tumor areas of RARb overexpressing xenografts. As for CA 19-9, increased CEA expression has been associated with increased tumor differentiation in a variety of studies.32,33 The intermediate filament Ck 7 belongs to the family of cytoskeletal proteins which, in the human pancreas, is exclusively expressed in the ductal cells.34 Previous studies have shown that Ck 7 expression is preserved in well-differentiated pancreatic ductal carcinoma cells / 5e20$$0042
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and lost in poorly differentiated carcinomas.35 Therefore, Ck 7 can be used as a valuable marker of duct cell differentiation. Although we observed only very few single cells staining positive for Ck 7 in control tumors, in the nonproliferating tumor areas of RARb overexpressing xenografts the majority of cells stained clearly positive for Ck 7. Negative staining for antibodies irrelevant for duct cell differentiation confirmed the specificity for the observed expression of duct cell specific differentiation markers. By conventional morphological criteria the development of duct-like structures is considered as a parameter for induction of differentiation in ductal pancreatic carcinoma cells. Overexpression of RARb in xenotransplanted DAN-G cells resulted in the appearance of cystic structures but not bona fide ducts. Several alternative hypotheses might explain this phenomenon. First, we intentionally chose the highly dedifferentiated cell line DAN-G for these experiments because of its very low level of RARb expression. Xenotransplants derived from this cell line display morphologically a highly dedifferentiated phenotype which, probably caused by multiple coexisting genetic alterations (see below), precludes complete redifferentiation by RARb expression into classical ductlike structures. Alternatively, induction of differentiation as suggested by biochemical markers in immunohistochemistry might precede morphological criteria of differentiation. Based on tumor burden, the animals had to be killed on day 18 postinjection for ethical reasons. Currently, we cannot exclude that induction of bona fide duct structures might have evolved at later time points. However, inhibition of growth, which is considered a hallmark of differentiation, as well as overexpression of four independent cellular molecules tightly associated with a more differentiated phenotype (CEA, CA 19-9, Ck 7, and MUC1) provide strong evidence that overexpression of RARb results in induction of differentiation in xenotransplanted DAN-G cells. Taken together, our in vivo data show that overexpression of RARb in DAN-G cells results in tumor areas, characterized by pronounced growth inhibition and induction of cellular differentiation compared with control tumors with neglectable RARb expression. Growth inhibition and induction of cellular differentiation could be observed in approximately 50% of the tumor areas at 18 days postinjection. This is most likely caused by the phenotype of the tumors generated from RARb transfected cells reflecting selective pressure against RARb expression.12 Furthermore, the selection pressure for RARb expression in transfected DAN-G cells, which is in vitro provided by culturing the cells in neomycin, cannot be maintained in vivo, which might result in WBS-Gastro
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selection of DAN-G cells which have lost the RARb expression in the absence of continuous selection pressure. Although restoration of RARb expression clearly shows beneficial therapeutic effects on the tumor biology of human pancreatic carcinomas, it can only partially reverse the malignant phenotype of pancreatic tumor cells. This can be explained by the many genetic and epigenetic changes occurring during malignant transformation in the pancreas, which act synergistically to release the cells from growth control mechanisms. Frequent genetic alterations observed in human pancreatic carcinomas are point mutations in the Ki-ras oncogene,36,37 overexpression of the c-myc oncogene,38 mutations in the adenomatous polyposis coli gene,39 as well as inactivation of the tumor-suppressor genes p53 and Rb-1.40,41 However, based on our previously reported beneficial effects of retinoids in pancreatic carcinoma,4 which are in large reproduced by RARb overexpression in this study, we believe that the potential therapeutic effects of RARb overexpression might bear important therapeutic consequences, such as the search of substances (e.g., hormones or cytokines) which can up-regulate RARb expression, the development of retinoid receptor specific ligands or the use of an RARb expression vector for gene therapy.
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References 1. Silverberg E, Boring CC, Squires TS. Cancer Statistics 1990. Can Cancer J Clin 1990;40:9–26. 2. Bakkevold KE, Arnesjo B, Kambestag B. Carcinoma of the pancreas and papilla of Vater: presenting symptoms, signs, and diagnosis related to stage and tumor site. A prospective multicentre trial in 472 patients. Scand J Gastroenterol 1992;27:317– 325. 3. Arbuck SG. Chemotherapy for pancreatic cancer. Baillieres Clin Gastroenterol 1990;4:953–964. 4. Rosewicz S, Stier U, Brembeck F, Kaiser A, Papadimitriou CA, Berdel WE, Wiedenmann B, Riecken EO. Retinoids: effects on growth, differentiation and nuclear receptor expression in human pancreatic carcinoma cell lines. Gastroenterology 1995;109: 1646–1660. 5. Petkovich M, Brand NJ, Krust A, Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 1987;330:444–450. 6. Zelent A, Krust A, Petkovich M, Kastner P, Chambon P. Cloning of murine and retinoic acid receptors and a novel receptor predominantly expressed in the skin. Nature 1989;339:714–717. 7. Heyman RA, Mangelsdorf DJ, Dyck JA, Stein RB, Eichele G, Evans RM, Thaller C. 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 1992;68:397–406. 8. Leid M, Kastner P, Chambon P. Multiplicity generates diversity in the retinoic acid pathway. Trends Biol Sci 1992;17:427–433. 9. Giguere V. Retinoic Acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr Rev 1994;15:61–79. 10. Hashimoto Y. Retinobenzoic acids and nuclear retinoic acid receptors. Cell Struct Funct 1991;16:113–123. 11. Dolle P, Ruberte E, Leroy P, Morriss-Kay G, Chambon P. Retinoic acid receptors and cellular retinoid binding proteins. A systematic
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30. Metzgar RS, Hollingsworth MA, Kaufman B. Pancreatic mucins. In: Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, Scheele GA, eds. The pancreas. New York: Raven, 1993:351– 367. 31. Dexter DL, Hager JD. Maturation induction of tumor cells using a human colon carcinoma mode. Cancer 1980;45:1178–1181. 32. Denk H, Tappeiner G, Eckerudorfer R, Holzner JH. Carcinoembryonic antigen (CEA) in gastrointestinal and extragastrointestinal tumors and its relationship to tumor cell differentiation. Int J Cancer 1972;10:262–272. 33. El-Deriny SE, O’Brien MJ, Christensen TG, Kupchik HZ. Ultrastructural differentiation and CEA expression of butyrate-treated human pancreatic carcinoma cells. Pancreas 1987;2:25–33. 34. Moll R, Krepler R, Franke WW. Complex cytokeratin polypeptide patterns observed in certain human carcinomas. Differentiation 1983;23:256–269. 35. Rafiee P, Ho SB, Bresalier RS, Bloom EJ, Kim JH, Kim YS. Characterization of the cytokeratins of human colonic, pancreatic, and gastric adenocarcinoma cell lines. Pancreas 1992;7:123–131. 36. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549–554. 37. Hruban RH, van Mansfeld ADM, Offerhaus GJA, van Weering DHJ, Allison DC, Goodman SN, Kensler TW, Bose KK, Cameron JL, Bos JL. K-ras oncogene activation in adenocarcinoma of the human pancreas. Am J Pathol 1993;143:545–554.
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38. Yamada H, Sakamoto H, Taira M, Nishimura S, Shomosato Y, Terada M, Sugimura T. Amplification of both c-Ki-ras with a point mutation and c-myc in a primary pancreatic cancer and its metastatic tumors in lymph nodes. Jpn J Cancer Res 1986;77:370– 375. 39. Horii A, Nakaturu S, Miyoshi Y, Ichii S, Nagase H, Ando H, Yanagisawa A, Tsuchiya E, Kato Y, Nakamura Y. Frequent somatic mutations of the APC gene in human pancreatic cancer. Cancer Res 1992;52:696–698. 40. DiGuiseppe JA, Hruban RH, Goodman SN, Allison DA, Cameron JL, Offerhaus JGA. Overexpression of p53 protein in adenocarcinoma of the pancreas. Am J Clin Pathol 1994;101:684–688. 41. Ruggeri B, Zhang SY, Caamano J, DiRado M, Flynn SD, KleinSzanto AJP. Human pancreatic carcinomas and cell lines reveal frequent and multiple alterations in the p53 and Rb-1 tumor suppressor genes. Oncogene 1992;7:1503–1511. Received December 10, 1996. Accepted April 15, 1997. Address requests for reprints to: Stefan Rosewicz, M.D., Department of Gastroenterology, Medizinische Klinik und Poliklinik, Klinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Fax: (49) 30-8445-4141. Supported by a grant from the Deutsche Krebshilfe (10 0954-Ro2) and the Maria Sonnenfeld Geda¨chtnisstiftung. The authors thank Dr. P. Chambon for providing us with the RAR b plasmid and Hoffmann-LaRoche for retinoids.
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Retinoic Acid Receptor b Regulates Growth and Differentiation in Human Pancreatic Carcinoma Cells ASTRID KAISER,* HERMANN HERBST,‡ GARY FISHER,§ MICHAEL KOENIGSMANN,\ WOLFGANG E. BERDEL,‡ ERNST–OTTO RIECKEN,* and STEFAN ROSEWICZ* Departments of *Gastroenterology, ‡Pathology and \Hematology/Oncology, Klinikum Benjamin Franklin, Berlin, Germany; and §Department of Dermatology, University of Michigan, Ann Arbor, Michigan
Background & Aims: Retinoic acid receptor b (RARb) expression is lost or decreased during malignant transformation in human pancreatic adenocarcinoma. The aim of this study was to evaluate the role of RARb expression in the propagation of a malignant phenotype in human pancreatic carcinoma cells. Methods: Overexpression of RARb in the human pancreatic carcinoma cell line DAN-G was achieved by selecting stable transfected cell clones. Genomic integration and expression were verified by Southern and Northern blotting and electrophoretic mobility shift assays. Growth was determined by cell number and xenografts transplanted into nude mice. Differentiation was examined by immunohistochemistry. Results: Overexpression of RARb in DAN-G cells inhibited cellular proliferation in vitro and in vivo. Furthermore, RARb overexpression resulted in induction of cellular differentiation in xenografted tumors as evidenced by increased tumor cell expression of duct cell differentiation markers carcinoembryonic antigen (CEA), CA19-9, and cytokeratin 7. Conclusions: Decreased expression of RARb plays a key role in the maintenance of a malignant phenotype in human pancreatic adenocarcinoma and therefore represents a novel target for experimental strategies in the treatment of pancreatic cancer patients.
A
denocarcinoma of the exocrine pancreas accounts for 25% of all gastrointestinal tumors and currently ranks as the fifth most common cause of cancer-related deaths in Western countries.1 Increasing incidence and lack of effective treatment options have resulted in a widespread therapeutic nihilism. Operative resection represents the only curative approach; however, at the time of diagnosis more than 90% of pancreatic cancer patients present with advanced regional disease or distant metastasis and can therefore not be considered for surgery.2 Conventional chemotherapy has not resulted in a significant survival benefit over the last 30 years, and the average survival time is still measured in months with a 5year survival rate of õ 2%.3 Based on these observations, it becomes clear that only a more detailed understanding of the tumor biology of pancreatic cancer will reveal novel / 5e20$$0042
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cellular targets for experimental therapeutic strategies. In this context, we have previously observed that natural and synthetic derivatives of vitamin A, biochemically summarized as retinoids, might offer a therapeutic alternative in pancreatic cancer patients. This is based on experimental evidence showing that retinoid treatment of human pancreatic carcinoma cells in vitro and in vivo results in a profound inhibition of tumor cell proliferation and induction of tumor cell differentiation.4 Retinoids exert these intriguing effects through interaction with specific nuclear receptors. Based on molecular cloning studies to date, two families of nuclear retinoid receptors have been described, each consisting of three receptor subtypes, a, b, and g: the retinoic acid receptors (RAR) which bind the naturally occurring retinoid all-trans retinoic acid (ATRA) with high affinity5,6; and the retinoid X receptors (RXR) whose naturally occurring, biologically active ligand is 9-cis retinoic acid, a geometric isomer of ATRA.7 The ligand-binding domains between these two retinoid receptor families share only a 29% sequence homology.8 In addition, each RAR/RXR gene generates multiple isoforms by either differential use of internal promotors or alternative splicing of exons.9 Both receptor families act as ligand dependent transcription factors, controlling gene transcription initiated from promotors of retinoid regulated genes by interacting with cis-acting DNA elements, the so-called retinoic acid responsive elements (RAREs).9,10 This multiplicity of receptors and gene pathways explains the diverse effects of retinoids on a wide range of cellular processes. Tissue-specific restricted expression of RAR/RXR subtypes and isoforms during embryogenesis and in the adult organism suggests that each RAR and RXR subtype exerts a unique biological function.11 Furthermore, Abbreviations used in this paper: ATRA, all-trans retinoic acid; bp, base pair; CEA, carcinoembryonic antigen; Ck, cytokeratin; CMV, cytomegalovirus; FCS, fetal calf serum; RAR, retinoic acid receptor; RARE, retinoic acid responsive elements; RT-PCR, reverse-transcription polymerase chain reaction; RXR, retinoid X receptors. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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disruption of the intracellular retinoid pathway via interference with specific RAR subtypes can result in carcinogenesis.12 – 14 Summarized with our observation that retinoids exert antiproliferative effects and induce differentiation in human pancreatic carcinoma cells4 we hypothesized that a deregulation in RAR subtype expression might be involved in the propagation of the malignant phenotype of human pancreatic cancer. In a subsequent in situ hybridization study, we analyzed RAR subtype expression in human pancreatic carcinoma tissue of 24 patients.15 Although there was no difference in the expression of RAR a and g between normal and malignant tissue, we observed that approximately one third of all pancreatic tumors completely lost the expression of RARb when compared with their nontransformed counterparts. Furthermore, the remaining tumors expressed significantly less RARb messenger RNA (mRNA) transcripts than adjacent normal pancreatic ductal cells. Moreover, we observed a tight correlation between the loss of RARb expression and the degree of cellular dedifferentiation.15 These data suggested that loss or decreased expression of RARb could either be an innocent epiphenomenon associated with malignant transformation or might indeed play a central role in the propagation of the malignant phenotype in human pancreatic adenocarcinoma. To dissect this problem we chose a reverse approach by overexpressing RARb in the human pancreatic tumor cell line DAN-G. We now show that restoration of RARb expression in human pancreatic tumor cells results in growth inhibition and induction of cellular differentiation.
Materials and Methods Materials The human pancreatic carcinoma cell line DAN-G was obtained from Deutsche Krebsforschungszentrum (Heidelberg, Germany). RPMI medium was obtained from GIBCO (Berlin, Germany), and charcoal-stripped, heat-inactivated fetal calf serum (FCS) from Biochrom (Berlin, Germany). All retinoids were provided by Hoffmann-LaRoche (Basel, Switzerland). We obtained [a-32P]-deoxycytidine triphosphate (6000 Ci/mmol) from DuPont (Bad Homburg, Germany). Random priming labeling kit and HybondN/ were obtained from Amersham (Braunschweig, Germany); nitrocellulose and blotting paper were purchased from Schleicher & Schuell (Dassel, Germany); Poly AT Tract isolation kit was obtained from Serva (Heidelberg, Germany); RNA and DNA molecular size markers, G418, and proteinase K were obtained from Bethesda Research Laboratories (BRL, Bethesda, MD); restriction enzymes from Boehringer Mannheim (Mannheim, Germany). The pRC-CMV mammalian expression vector was obtained from Invitrogen (La Jolla, CA). All other chemicals were of analytical grade and purchased from Sigma (Deisenhofen, Germany).
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Cell Culture and Growth Assays DAN-G cells were grown as subconfluent monolayer cultures supplemented with 10% (vol/vol) charcoal-stripped, heat-inactivated FCS penicillin (100 U/mL), and streptomycin (100 mg/mL). The cells were maintained under 95% air and 5% CO2 at 377C. All experiments were performed in the log phase of growth after the cells had been plated for 24 hours unless otherwise stated. Cell viability was routinely checked by trypan blue exclusion test and was consistently found to be ú95%. After mild trypsinization, cells were plated in 22 mm culture dishes at a density of 50,000 cells/well in the presence of RPMI containing 10% FCS. At the indicated times, cells were washed with 154 mmol/L NaCl and then obtained by trypsinization. Cells were then resuspended in phosphate-buffered saline to ensure a single cell suspension. Viable cells were counted in a hemocytometer by trypan blue exclusion. Triplicate wells were analyzed for each time point.
Stable Transfection of DAN-G Cells The 1.5-kilobase (kb) complementary DNA (cDNA) of human RAR-b2 (formerly described as RAR-b06) was isolated from the host vector pSG5 using SacI and BamHI restriction digests and subcloned in the linearized HindIII site of pRC/ CMV vector, containing a neomycin resistance gene. This yielded the RAR-b2 cDNA preceded by the cytomegalovirus (CMV) promotor and followed by the human growth hormone termination and polyadenylation signal. The construct was confirmed by restriction analysis. DAN-G cells, (approximately 4 1 106 cells per 100 mm dish) were transfected with 5 mg of pRC-b2 plasmid using the Lipofectamine Reagent (GIBCO BRL, Bethesda, MD) following the instructions supplied by the manufacturer. Control cells were transfected with vector alone (mock-transfected) not containing RARb-specific nucleotide sequences. Twenty-four hours after transfection, cells were diluted 1:10 and plated in medium containing 1 mg/mL G418. Resistant cell clones appeared after approximately 21 days and were picked for expansion at 35 days. The RARb and mock-transfected cell clones were maintained under selection pressure for all the experiments.
Southern Blotting For isolation of genomic DNA from transfected and parental cells, 3–4 1 107 cells were collected by trypsinization and centrifugation. The pellet was resuspended in 100 mmol/ L NaCl, 10 mmol/L Tris-HCI pH 8, 25 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate, and 1 mg/mL proteinase K and incubated with shaking at 507C for 12 hours. Samples were extracted twice with an equal volume of phenol/chloroform/isoamylalcohol (vol/vol 25:24:1) and treated with ribonuclease A (10 mg/mL) at 377C for 4 hours. DNA was precipitated with 1/10 volume sodium acetate and ethanol at room temperature. After low-speed centrifugation the pellet was rinsed with 70% ethanol. Fifty micrograms of genomic DNA were digested with EcoRI and separated on a 1% agarose gel at 47C overnight. After denaturation, DNA
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was transferred to HybondN/ and prehybridization and hybridization was performed exactly as previously described.4
RNA Isolation and Northern Blotting RNA was isolated by a modification of the method of Chirgwin et al16 exactly as previously described.4 Poly(A)/purified RNA was isolated using the Poly AtTract kit following the instructions supplied by the manufacturer. Qualitative analysis of RNA was performed using Northern blot analysis exactly as previously described.4 RNA was denatured by formaldehyde, subjected to electrophoresis through a 1% agarose gel in the presence of formaldehyde, and then transferred to nitrocellulose. An RNA ladder was electrophoresed in parallel for size determination. The RARb cDNA insert was electroeluted and then radioactively labeled using the random primer labeling kit following the instructions provided by the supplier. Unincorporated nucleotides were removed by passing the reaction sample over a Sephadex G 50 column (Sigma, Deisenhofen, Germany). The specific activity of the probes was routinely 1–2 1 109 cpm/ mg DNA. Filters were prehybridized and hybridized exactly as previously described.4 After hybridization, filters were sequentially washed with increasing stringency as previously described.4 Filters were then blotted dry and exposed to x-ray film for 2 days using two intensifying screens.
Electrophoretic Mobility Shift Assays Nuclear extracts from DAN-G cells were prepared by using a micropreparation technique. Five micrograms of nuclear protein extracts were incubated on ice for 15 minutes with 2 mg of poly[d(I-C)]/poly [(I-C)] in 20 mmol/L TrisHCL pH 7.9, 1 mmol/L EDTA, 0.1% NP-40, 1 mmol/L dithiotreitol, 6.25% Ficoll, and 0.1 mg/mL bovine serum albumin (BSA) in a final reaction volume of 25 mL. A doublestranded oligideoxynucleotide (5*-TCGACTAAGGGTTCACCGAAAGTTCACTCGCA-3*) containing the retinoic acid response element in the RARb2 promotor was used as a probe. The oligonucleotide was end-labeled with [g-32P]ATP by a T4 polynucleotide kinase reaction, and unincorporated nucleotides were removed by passing the reaction mixture over a DE52 anion exchange column (Whatman, Maidstone, England). We added 0.1 ng radioactively labeled oligonucleotide to each reaction and incubated for 30 minutes at room temperature. For competition experiments a 40-fold excess of unlabeled oligonucleotide was added to the reaction mixture before the addition of labeled oligonucleotide. The samples were electrophoresed on a 6% polyacrylamide gel. Radioactive proteinDNA complexes in the gels were quantitated using a PhosphorImager (Berthold, Berlin, Germany).
Tumor Growth in Nude Mice Four-week-old female athymic BALB/c nu/nu mice were obtained from Bomholtgard Ltd. (Ry, Denmark). Mice were maintained under specific pathogen–free conditions and fed an autoclaved standard diet (Altromin 1324; Altromin, Lage, Germany) and sterilized water at low pH. Human pancreatic tumor cells were xenotransplanted intracutaneously into
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the flank as single cell suspensions in 0.1 mL phosphate-buffered saline. Equal amounts (5 1 105) of viable wild-type and RARb transfected cells were injected into each animal. Tumor growth was measured with calipers at days as indicated. Mean diameter (length plus width/2) is given as tumor size in millimeters.
Histological Analysis of Xenografts Tumors were immediately removed after killing the animals. Four-micrometer serial sections of formol-fixed/paraffin-embedded tissue blocks mounted on 3-aminopropylethoxysilane-treated slides were stained by the immunoalkaline phosphatase method.17 Affinity-purified rabbit anti-mouse immunoglobulin antibodies and immunoalkaline phosphatase complex (diluted 1:20) were obtained from DAKO (Glostrup, Denmark). Throughout these studies, controls consisted of substitution of the primary antibody by normal serum. The following primary antibodies were used for immunohistochemistry: monoclonal antibody against carcinoembryonic antigen (CEA)18 obtained from DAKO (Glostrup, Denmark) and monoclonal antibody against cytokeratin 719 obtained from Boehringer Mannheim (Mannheim, Germany). Monoclonal antibodies specific for the carbohydrate antigen CA19-920 and for the proliferation-associated antigen Ki-67 (clone mib-1)21 were from CIS Diagnostik (Dreieich, Germany) and Dianova (Hamburg, Germany), respectively.
Statistics Results were evaluated by repeated measurement twoway analysis of variance with a multiple contrast test (Hotelling T2). P values of õ0.05 were considered to be significant.
Results Generation of a RARb Overexpressing DAN-G Cell Clone Integration of transfected RARb DNA into the genome after stable transfection was investigated by Southern blotting. As shown in Figure 1, probing the EcoRI-digested genomic DNA from the wild-type parental, mock-transfected and RARb transfected cells with a RARb cDNA, revealed an identical fragment hybridization pattern corresponding to the endogenous RARb gene in all cell lines. Only RARb transfected cells however showed additional bands of 1300 base pairs (bp), 610 bp, and 190 bp, which had to be expected from the internal EcoRI restriction fragment size of the transfected RARb cDNA. Thus, a DAN-G cell clone was established with stable integration of the RARb in its genome. We then used Northern blot analysis to investigate mRNA transcription from the integrated DNA. Hybridization with the RARb cDNA showed a strong signal of approximately 2.0 kilobases in the RARb transfected DAN-G cell line, which corresponds to the expected size of the transcribed mRNA for RARb2 WBS-Gastro
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(Figure 2). In contrast, no significant hybridization signal for RARb could be observed in the parental wildtype and mock-transfected DAN-G cells (Figure 2). This is in agreement with our previous observation that parental DAN-G cells express trace amounts of RARb mRNA, which can only be detected by the highly sensitive reverse-transcriptase polymerase chain reaction (RT-PCR) technique but can barely be detected by conventional Northern blotting using poly(A)/ purified RNA.4 Transfection of RARb cDNA had no effect on RARa and RARg mRNA expression when compared with mock-transfected controls or wild-type cells (data
not shown). Therefore, this DAN-G cell clone shows selective overexpression of RARb mRNA compared with the parental wild-type and mock-transfected controls. Of the initially selected 4 cell clones, 3 of 4 revealed genomic integration of RARb but only 1 of 4 clones showed appreciable RARb mRNA overexpression. This cell clone was used for all further studies. To verify that the transfected RARb mRNA is translated into a functionally active protein, we performed electrophoretic mobility shift assays using a RARE in the RARb2 promotor as a probe. As seen in Figure 3, RARb transfected DAN-G cells display a 4–5-fold stronger RARE binding as compared with wild-type or mock-transfected controls, because of overexpression of a functionally active RARb protein. Effects of RARb Overexpression on DAN-G Cells In Vitro We next investigated the effects of RARb transfection on cellular proliferation in DAN-G cells. Overexpression of RARb results in a significant decrease of tumor cell growth compared with mock-transfected or untransfected control cells (Figure 4). This inhibition
Figure 1. Genomic integration of RARb in transfected DAN-G cells. After stable transfection, EcoRI-digested DNA from wild-type, mocktransfected, and RARb transfected DAN-G cells was analyzed by Southern blotting using the radioactively labeled cDNA probe for RARb2 . The expected fragment sizes are deduced from the internal EcoRI sites (E) of the transfected RARb nucleotide sequence (190 and 610 bp). The 1300-bp fragment corresponds to the next EcoRI restriction site in the DAN-G cell genome. A shorter exposition of the autoradiograph revealed a 1200-bp fragment size corresponding to the endogenous and a 1300-bp fragment size corresponding to the transfected RARb. A longer exposition of the autoradiograph had to be chosen to visualize the weaker 190-bp fragment. Molecular size determination was obtained from a DNA ladder that was electrophoresed in parallel. The somewhat weaker hybridization signal in mocktransfected DAN-G cells is because of a lesser amount of digested DNA as evidenced from the ethidium bromide stain of the gel. Shown is a representative of two independent experiments.
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Figure 2. Expression of RARb mRNA in transfected DAN-G cells. Twenty micrograms of poly(A)/ purified RNA from wild-type, mocktransfected, and RARb transfected DAN-G cells were separated electrophoretically and then analyzed by Northern blotting using the random primed cDNA probe for RARb. Molecular size was determined by an RNA ladder, electrophoresed in parallel. Only RARb transfected DAN-G cells expressed a clearly visible 2.0-kb mRNA corresponding to RARb. Shown is a representative of two independent expriments, yielding identical results.
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Effects of RARb Overexpression on DAN-G Cells In Vivo
Figure 3. Electrophoretic mobility shift assay of RARb transfected DAN-G cells. EMSAs using a double-stranded oligonucleotide containing the RARE of the RARb2 promotor were performed in the presence (/) or absence (0) of a 40-fold excess of cold oligonucleotide. RARb overexpressing DAN-G cells display an increased binding of nuclear extract proteins to the RARE oligonucleotide compared with mock-transfected and wild-type controls. Shown is a representative of three independent experiments, yielding almost identical results.
of growth in the absence of endogenous (charcoalstripped, heat-inactivated FCS) and exogenous retinoids could be observed as early as 48 hours and remained significant for up to the investigated time period of 5 days. In contrast, transfection of DAN-G cells with plasmid alone, devoid of RARb nucleotide sequence (mock-transfected), did not result in growth inhibition compared with the parental wild-type, indicating that overexpression of RARb is specifically responsible for the observed growth retardation. Furthermore, overexpression of RARb had no significant effect on the growth inhibition mediated by ATRA when compared with mock-transfected or wild-type DANG cells (cell numbers after 120 hours of 10 mmol/L ATRA were 62% { 5% [n Å 5] and 67% { 8% [n Å 6] of untreated controls for mock-transfected and RARb transfected, respectively). / 5e20$$0042
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Subsequently, we have evaluated putative tumor growth retardation by RARb overexpression in xenografts of DAN-G cells in nu/nu BALB/c mice in vivo. Preliminary experiments showed a reliable and reproducible tumor formation using this cell line at a cell load of 5 1 105 cells per mouse. For these experiments we compared wild-type and RARb transfected cells, because in the previous experiments no difference in terms of growth characteristics and morphological changes were observed between mock-transfected and wild-type DANG cells. We observed some remarkable differences between the two experimental groups. For one, nude mice injected with wild-type DAN-G cells showed a rather rapidly developing cachexia as compared with the animals bearing RARb transfected DAN-G tumors. This observation was reflected by whole animal weight determined 15 days after tumor cell injection, which was 15.61 { 3.15 g (n Å 10) in wild-type injected versus 18.71 { 1.21 g (n Å 11) in animals that had been injected with RARb overexpressing DAN-G cells. However, these differences did not reach statistical significance. In accordance with our in vitro data, we observed a significant growth retardation of RARb transfected DAN-G cell xenograft tumors compared with the untransfected controls (Figure 5), the differences becoming statistically significant at day 13 after the injection. Effects of RARb Overexpression on Tumor Cell Proliferation and Differentiation In Vivo Histological analysis of the tumors showed the appearence of a dedifferentiated adenocarcinoma with a
Figure 4. Effects of RARb overexpression on DAN-G cell proliferation in vitro. DAN-G cells (mock-transfected and RARb transfected) were plated at a density of 50,000 cells per well in triplicates. Viable cell number was determined by counting cells after trypan blue exclusion at the indicated time periods. Cell viability ú 95% was required before the experiment was evaluated. The growth curve of mock-transfected and parental DAN-G cells was identical. Shown is the MEAN { SEM of five independent experiments, each performed in triplicate (*P õ 0.05).
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the mucin carbohydrate epitope Ca19-9 showed a moderately strong and diffuse staining pattern in control tumors (Figure 7C). However, in RARb overexpressing xenografts a very strong Ca19-9 expression could be detected in the nonproliferating, CEA-expressing cystic tumor areas (Figure 7D). The intermediate filament protein cytokeratin 7 (Ck7) was only expressed sporadically in very few single cells of nontransfected control tumors (Figure 7E), whereas we observed significantly increased expression in the areas of presumed differentiation (Figure 8F). Furthermore, we observed a restricted overexpression of another ductal epithelial differentiation marker, the mucin MUC-1 in the nonproliferating cystic areas of RARb transfected xenografts, basically following the expression pattern of CEA (data not shown). Control
Figure 5. Effects of RARb overexpression on tumor growth in vivo. Xenograft tumor growth of wild-type and RARb transfected DAN-G cells was monitored at the indicated time points as outlined in Materials and Methods. Shown are the MEAN { SEM of 10 (wild-type DAN-G cells) and 11 (RARb transfected DAN-G cells) animals (*P õ 0.05).
modest stromal reaction (Figure 6). No major infiltration with hematopoietic or lymphocytic murine cells was observed in both groups (Figure 6). In comparison to the control tumors, we observed multiple cystic structures containing aggregates of tumor cells in the RARb transfected xenografts (Figure 6C and D). We then determined the fraction of actively proliferating tumor cells by immunohistochemistry using the mib-1 antibody directed against the proliferation-associated antigen Ki67.21 In control tumors, more than 90% of all tumor cells stained positively for mib-1, indicating a highly aggressive tumor biology (Figure 6E); in contrast, the fraction of proliferating cells in RARb transfected xenografts was remarkably reduced to approximately 50%, showing virtually no staining in the cystic structures, which were exclusively noted in the RARb transfected xenografts (Figure 6F). To investigate whether the inhibition of growth in RARb transfected xenografts was paralleled by induction of cellular differentiation, we performed immunohistochemistry with a panel of antibodies directed against molecules, commonly associated with increased duct cell differentiation. CEA was expressed at barely detectable concentrations in control tumors (Figure 7A). However, in RARb transfected xenografts, we observed a very strong CEA expression, which was focused and mainly restricted to the cystic nonproliferating areas, shown to stain negative for mib-1 (Figure 7B). Antibodies against / 5e20$$0042
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Figure 6. Effects of RARb overexpression on tumor morphology. Histological analyses and immunohistochemistry of (A, C, and E ) control tumors and (B, D, and F ) RARb overexpressing xenografts were performed as outlined in Materials and Methods. (A–D) H&E staining of tumor sections revealed the appearence of multiple cystic areas with tumor cell aggregrates (arrows in B and F ), which were only observed in RARb overexpressing xenografts. (A and C ) Control tumors displayed a solid dedifferentiated adenocarcinoma. Immunohistochemistry using the monoclonal antibody mib-1, directed against the proliferation associated Ki-67 antigen, revealed almost homogenous tumor cell staining in control tumors (E ), whereas the cystic tumor areas in RARb overexpressing xenografts were predominantly negative (delineated by arrows in F ). Shown is a representative analysis of 4 animals for each experimental group, which all revealed similar results (original magnification: A and B, 661; C and D, 1321; E and F, 661).
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Figure 7. Effects of RARb overexpression on tumor differentiation. Immunohistochemistry using monoclonal antibodies against (A and B ) CEA, (C and D ) CA 19-9, (E and F ) and cytokeratin 7 (CK 7) of (A, C, and E ) control tumors and (B, D, and F ) RARb overexpressing xenografts was performed as outlined in Materials and Methods. Shown is the pronounced increase in the expression of CEA, CA 199 and Ck 7 in the cystic, nonproliferating tumor areas of RARb overexpressing xenografts (arrows in B, D, and F ). Ck 7 stained only very few single cells in control tumors (arrow in E ). Shown is a representative of four animals for each experimental group, all yielding very similar results. (Original magnification: A-B, 661; C-D, 131; E-F, 661.)
antibodies irrelevant for pancreatic duct cell differentiation (Ck 20 and synaptophysin) stained consistently negative in control and RARb transfected tumors, confirming the specificity of the observed expression for the differentiation associated markers. Taken together, these immunohistochemical data suggest that overexpression of RARb in DAN-G xenografts results in multiple focal areas of tumor growth inhibition and induction of cellular differentiation.
Discussion We have previously shown that retinoids inhibit tumor cell proliferation and induce cellular differentiation in human pancreatic carcinoma cells in vitro and in vivo.4 In addition, we observed that RARb expression is either lost or significantly decreased in human pancreatic carcinomas when compared with the nontransformed human pancreas.15 This observation is reflected by in vitro / 5e20$$0042
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experiments, showing a significantly higher RARb expression in the well-differentiated human pancreatic tumor cell line Capan 122 (grade I according to the grading system by Kern et al.23) compared with the poorly differentiated tumor cell line DAN-G (grade III), in which RARb is only expressed at very low levels and exclusively detectable by the highly sensitive RT-PCR technique.4 This tight correlation between cellular differentiation and RARb expression in vivo and in vitro suggested that RARb might play an important role in the propagation of the malignant phenotype in human pancreatic adenocarcinoma. To investigate this hypothesis, we conferred RARb overexpression to the poorly differentiated human pancreatic tumor cell line DAN-G and examined the consequences in respect to tumor biology of this cell line. The RARb gene consists of four isoforms, generated by differential splicing, as well as differential use of promotors.24 In humans, the RARb3 equivalent does not appear to exist and RARb1 expression is principally limited to fetal cells and small cell lung cancer cells.12 The RARb4 isoform has been shown to act as a tumor promoter in lung and breast tissue of transgenic mice25 and is not expressed in human pancreatic carcinoma cell lines.4 We therefore decided to overexpress the RARb2 isoform, which also is the predominant isoform expressed in human pancreatic carcinoma cells.4 Selection of DAN-G cells, stably transfected with the RARb2 cDNA under control of the CMV promotor, showed one cell clone with stable integration of RARb nucleotide sequence in its genome and considerable overexpression of RARb mRNA transcripts and protein as judged by Southern and Northern blot analysis as well as electrophoretic mobility shift assays. Overexpression of RARb resulted in growth inhibition of DAN-G cells in vitro. This antiproliferative effect could be observed in the absence of biologically relevant retinoid concentrations (heat-inactivated, charcoalstripped FCS contains õ10013 mol/L ATRA). This observation confirms recent studies, suggesting that ligand occupancy is not always necessarily required for retinoid receptor-mediated transcriptional activation12,26 and that, for example, phosphorylation alone (e.g., caused by the addition of serum) can activate retinoid-receptor mediated transcriptional regulation.27 This is further supported by RARb overexpression experiments in human epidermoid lung cancer cells, showing that RARb overexpression alone, in the absence of retinoids, can induce antiproliferative effects.12 One possible explanation for this phenomenon is based on the observation that RAR can form a nonproductive complex with the proliferation associated AP-1 transcription factor, constituted by c-Jun homodimers or c-Jun/c-Fos heterodimWBS-Gastro
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ers.28,29 This experimental evidence provides a simple mechanism by which the intracellular concentration of RAR might limit cell growth and malignant progression. Whether phosphorylation or RARb/AP-1 interaction is indeed responsible for the observed growth inhibition in RARb overexpressing DAN-G cells is currently the subject of further investigation. When xenografted into nude mice, RARb overexpressing DAN-G tumor-bearing animals did not suffer to the same extent from the pronounced weight loss and cachexia observed in the control animals. We currently do not understand the molecular basis for this observation. However, it seems conceivable that RARb overexpression, which results in inhibition of growth and induction of cellular differentiation in vivo, mediates decreased expression of systemic acting factor(s) synthesized in the tumor cell, which might be responsible for the observed tumor-associated cachexia. In analogy to the in vitro experiments, we observed a significantly decreased growth rate of RARb overexpressing DAN-G cell tumors when xenografted in nude mice. Histological analysis showed multiple areas with a cystic appearence of tumor cell aggregates in RARb overexpressing tumors; immunohistochemistry, using an antibody against the proliferation-associated antigen Ki67, showed that the majority of these cells were not actively proliferating, compared with the control tumors where virtually more than 90% of all tumor cells stained positive for Ki-67. Given that inhibition of growth represents a hallmark of cellular differentiation, we investigated the differentiation state of these nonproliferating tumor cells using a panel of antibodies directed against antigens commonly associated with cellular differentiation of pancreatic carcinomas. The mucin carbohydrate epitope detected by monoclonal antibody Ca19-9, which is also expressed in normal ductal epithelium, was considerably stronger expressed in the nonproliferating tumor cell areas of RARb overexpressing xenografts. Expression of this antigen is commonly associated with well-differentiated pancreatic tumors and either only sporadically expressed or absent in poorly differentiated carcinomas.30,31 CEA, which was barely detectable in control tumors, was also very strongly expressed in the nonproliferating cystic tumor areas of RARb overexpressing xenografts. As for CA 19-9, increased CEA expression has been associated with increased tumor differentiation in a variety of studies.32,33 The intermediate filament Ck 7 belongs to the family of cytoskeletal proteins which, in the human pancreas, is exclusively expressed in the ductal cells.34 Previous studies have shown that Ck 7 expression is preserved in well-differentiated pancreatic ductal carcinoma cells / 5e20$$0042
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and lost in poorly differentiated carcinomas.35 Therefore, Ck 7 can be used as a valuable marker of duct cell differentiation. Although we observed only very few single cells staining positive for Ck 7 in control tumors, in the nonproliferating tumor areas of RARb overexpressing xenografts the majority of cells stained clearly positive for Ck 7. Negative staining for antibodies irrelevant for duct cell differentiation confirmed the specificity for the observed expression of duct cell specific differentiation markers. By conventional morphological criteria the development of duct-like structures is considered as a parameter for induction of differentiation in ductal pancreatic carcinoma cells. Overexpression of RARb in xenotransplanted DAN-G cells resulted in the appearance of cystic structures but not bona fide ducts. Several alternative hypotheses might explain this phenomenon. First, we intentionally chose the highly dedifferentiated cell line DAN-G for these experiments because of its very low level of RARb expression. Xenotransplants derived from this cell line display morphologically a highly dedifferentiated phenotype which, probably caused by multiple coexisting genetic alterations (see below), precludes complete redifferentiation by RARb expression into classical ductlike structures. Alternatively, induction of differentiation as suggested by biochemical markers in immunohistochemistry might precede morphological criteria of differentiation. Based on tumor burden, the animals had to be killed on day 18 postinjection for ethical reasons. Currently, we cannot exclude that induction of bona fide duct structures might have evolved at later time points. However, inhibition of growth, which is considered a hallmark of differentiation, as well as overexpression of four independent cellular molecules tightly associated with a more differentiated phenotype (CEA, CA 19-9, Ck 7, and MUC1) provide strong evidence that overexpression of RARb results in induction of differentiation in xenotransplanted DAN-G cells. Taken together, our in vivo data show that overexpression of RARb in DAN-G cells results in tumor areas, characterized by pronounced growth inhibition and induction of cellular differentiation compared with control tumors with neglectable RARb expression. Growth inhibition and induction of cellular differentiation could be observed in approximately 50% of the tumor areas at 18 days postinjection. This is most likely caused by the phenotype of the tumors generated from RARb transfected cells reflecting selective pressure against RARb expression.12 Furthermore, the selection pressure for RARb expression in transfected DAN-G cells, which is in vitro provided by culturing the cells in neomycin, cannot be maintained in vivo, which might result in WBS-Gastro
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selection of DAN-G cells which have lost the RARb expression in the absence of continuous selection pressure. Although restoration of RARb expression clearly shows beneficial therapeutic effects on the tumor biology of human pancreatic carcinomas, it can only partially reverse the malignant phenotype of pancreatic tumor cells. This can be explained by the many genetic and epigenetic changes occurring during malignant transformation in the pancreas, which act synergistically to release the cells from growth control mechanisms. Frequent genetic alterations observed in human pancreatic carcinomas are point mutations in the Ki-ras oncogene,36,37 overexpression of the c-myc oncogene,38 mutations in the adenomatous polyposis coli gene,39 as well as inactivation of the tumor-suppressor genes p53 and Rb-1.40,41 However, based on our previously reported beneficial effects of retinoids in pancreatic carcinoma,4 which are in large reproduced by RARb overexpression in this study, we believe that the potential therapeutic effects of RARb overexpression might bear important therapeutic consequences, such as the search of substances (e.g., hormones or cytokines) which can up-regulate RARb expression, the development of retinoid receptor specific ligands or the use of an RARb expression vector for gene therapy.
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30. Metzgar RS, Hollingsworth MA, Kaufman B. Pancreatic mucins. In: Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, Scheele GA, eds. The pancreas. New York: Raven, 1993:351– 367. 31. Dexter DL, Hager JD. Maturation induction of tumor cells using a human colon carcinoma mode. Cancer 1980;45:1178–1181. 32. Denk H, Tappeiner G, Eckerudorfer R, Holzner JH. Carcinoembryonic antigen (CEA) in gastrointestinal and extragastrointestinal tumors and its relationship to tumor cell differentiation. Int J Cancer 1972;10:262–272. 33. El-Deriny SE, O’Brien MJ, Christensen TG, Kupchik HZ. Ultrastructural differentiation and CEA expression of butyrate-treated human pancreatic carcinoma cells. Pancreas 1987;2:25–33. 34. Moll R, Krepler R, Franke WW. Complex cytokeratin polypeptide patterns observed in certain human carcinomas. Differentiation 1983;23:256–269. 35. Rafiee P, Ho SB, Bresalier RS, Bloom EJ, Kim JH, Kim YS. Characterization of the cytokeratins of human colonic, pancreatic, and gastric adenocarcinoma cell lines. Pancreas 1992;7:123–131. 36. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549–554. 37. Hruban RH, van Mansfeld ADM, Offerhaus GJA, van Weering DHJ, Allison DC, Goodman SN, Kensler TW, Bose KK, Cameron JL, Bos JL. K-ras oncogene activation in adenocarcinoma of the human pancreas. Am J Pathol 1993;143:545–554.
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38. Yamada H, Sakamoto H, Taira M, Nishimura S, Shomosato Y, Terada M, Sugimura T. Amplification of both c-Ki-ras with a point mutation and c-myc in a primary pancreatic cancer and its metastatic tumors in lymph nodes. Jpn J Cancer Res 1986;77:370– 375. 39. Horii A, Nakaturu S, Miyoshi Y, Ichii S, Nagase H, Ando H, Yanagisawa A, Tsuchiya E, Kato Y, Nakamura Y. Frequent somatic mutations of the APC gene in human pancreatic cancer. Cancer Res 1992;52:696–698. 40. DiGuiseppe JA, Hruban RH, Goodman SN, Allison DA, Cameron JL, Offerhaus JGA. Overexpression of p53 protein in adenocarcinoma of the pancreas. Am J Clin Pathol 1994;101:684–688. 41. Ruggeri B, Zhang SY, Caamano J, DiRado M, Flynn SD, KleinSzanto AJP. Human pancreatic carcinomas and cell lines reveal frequent and multiple alterations in the p53 and Rb-1 tumor suppressor genes. Oncogene 1992;7:1503–1511. Received December 10, 1996. Accepted April 15, 1997. Address requests for reprints to: Stefan Rosewicz, M.D., Department of Gastroenterology, Medizinische Klinik und Poliklinik, Klinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Fax: (49) 30-8445-4141. Supported by a grant from the Deutsche Krebshilfe (10 0954-Ro2) and the Maria Sonnenfeld Geda¨chtnisstiftung. The authors thank Dr. P. Chambon for providing us with the RAR b plasmid and Hoffmann-LaRoche for retinoids.
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