Pancreas Vol. 5 , No. 5 , pp. 507-514 0 1990 Raven Press, Ltd., New York
Carbonic Anhydrase I1 Gene Expression in Cell Lines from Human Pancreatic Adenocarcinoma "Marsha L. Frazier, ?Brenda J. Lilly, *Elsie F. Wu, TTatsuya Ota, and ?David Hewett-Emmett *Section of Gastrointestinal Oncology and Digestive Diseases, Department of Medical Oncology, The University of Texas, M.D. Anderson Cancer Center, and tGenetics Centers, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, U.S.A.
Summary: Current evidence suggests that carbonic anhydrase I1 (CA 11) is produced by pancreatic duct cells but not by pancreatic acinar or islet cells. The aim of this study was to determine whether CA I1 homologous RNA and CA I1 immunoreactive protein are produced by cell lines established from human pancreatic adenocarcinomas. A 1.7-Kb CA I1 homologous RNA was detected in poly(A+) RNA isolated from normal human pancreas, normal human liver, and to varying degrees in the cell lines examined. The CA I1 immunoreactivity corresponding to approximately 30 kD (consistent with the established molecular mass of CA 11) was also detected by immunoblotting in normal human pancreas, normal human liver, and some of the cell lines. We also found that the levels of CA I1 homologous RNA increase in the pancreatic adenocarcinoma cell lines following treatment with the differentiating agent, retinoic acid. Key Words: Carbonic anhydrase 11-Pancreatic adenocarcinoma-RNA-Retinoic acid.
types (2). A third form, CA 111, is abundant, and has been purified from the skeletal muscle of several mammalian species and extensively characterized (1). The CA IV, whose properties have been reviewed by Wistrand (3), is a membrane-associated form that has been purified from kidney brush border and lung membranes (4). The CA V was identified in liver mitochondria (5,6) and CA VI in human salivary glands (7). The CA VI from sheep salivary glands has been sequenced (8). A seventh gene (CA VII, formerly CA Z) is probably expressed in an as yet unidentified tissue (J. C. Montgomery, personal communication). This suggests that there may be as many as seven members of the CA gene family. Carbonic anhydrase activity was first detected in extracts of the pancreas by van Goor (9), who recognized the need to correct for red cell CA contam-
Carbonic anhydrases (CAs) (E.C. 4.2.1.1.) are zinc-containing metalloenzymes that catalyze the reversible hydration of carbon dioxide and are products of a multigene family. At least seven genetically different forms of the enzyme have been identified or inferred from gene sequences. In mammals, the two most widely expressed forms, CA I and CA 11, have different kinetic and antigenic properties. The CA I has low and CA I1 has high specific activity (1). Each is produced in erythrocytes, as well as in a variety of other tissues and cell Manuscript received May 8, 1989; revised manuscript accepted November 13, 1989. Address correspondence and reprint requests to Dr.Marsha L. Frazier, Section of Gastrointestinal Oncology and Digestive Diseases, Department of Medical Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, U.S.A.
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ination. The enzyme catalyzes the hydration of carbon dioxide and the dehydration of bicarbonate ions, thus enabling the duct cells of the pancreas to produce a bicarbonate-rich fluid containing the acinar cell-derived digestive enzymes. The fluid passes from the duct system into the duodenum, neutralizing the duodenal contents. Histochemical studies using antisera against CA I1 suggest that at least some of the pancreatic CA activity is due to CA 11and that duct cells but not acinar cells or islet cells are CA I1 positive (10). METHODS AND MATERIALS Tissue and cell lines Normal human pancreas and liver were obtained from autopsy. The following cell lines that were obtained from the American Type Culture Collection and used in this study were Capan-2, Mia PaCa-2, BxPC-3, and PANC-1. MDAPanc-3 was established here at the M.D. Anderson Cancer Center from a human liver metastasis of a pancreatic adenocarcinoma and has been described elsewhere (11). Retinoic acid (all trans) was used in some of these studies. The compound was prepared as a lo-* M stock solution in dimethyl sulfoxide (DMSO). To determine the level of growth inhibition of various concentrations of retinoic acid on the cell line BxPC-3, we seeded cells at 2 x 10’ cells per dish in a series of 35-mm-diameter dishes and incubated them at 37°C for 24 h in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. At day 0, the cultures were then replaced with fresh medium containing DMSO (control) at concentrations of 0.001, 0.01, or 0.1%, or retinoic acid or lo-’ M. The at concentrations of lo-’, cultures were re-fed on day 3 with retinoic acid containing medium. On day 5 , the cells were removed from the plates and counted. Cell growth inhibition was expressed as a percentage of the growth of the controls. Larger quantities of cells were grown in larger flasks and volumes of media. For time course studies, the cells were fed with fresh retinoic acid containing medium 76 h prior to each of the harvesting time points to minimize possible effects of culture feeding on the expression of CA 11 sequences. Preparation of the CA I1 cDNA probe The recombinant carbonic anhydrase pCA 38.3 probe that we utilized in these studies corresponds to a human CA I1 cDNA and has been described Pancreas, VoI. 5, No. 5, 1990
(12). It contains the entire coding region, 38 base pairs of the 5’ untranslated region and approximately 750 base pairs of the 3’ untranslated region. The total length of the fragment is 1.4 kilobase pairs (Kb). Extraction of RNA from normal human pancreas, liver, and cell lines Total RNA was isolated from the normal human pancreas and liver utilizing the proteinase K technique described by Frazier et al. (13). For tissue cultured cell lines, RNA was extracted from cell pellets. The pellets were prepared from cell monolayers covering approximately 90% of the area of the growing surface in the flask. The cells were scraped off using a disposable plastic conical centrifuge tube and centrifuged for 5 min at 1,000 rpm in a clinical centrifuge. The medium was aspirated, and the packed cell pellets were stored at -80°C until the RNA was extracted. Poly(A+) RNA was then isolated as described by Aviv and Leder (14). Blot analysis of Poly(A+) RNA Poly(A + ) RNA was fractionated by electrophoresis through 1.2% agarose gels containing 6% formaldehyde and transferred onto nitrocellulose filters, as described by Rave et al. (15). A 0.259.5-Kb RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD, U.S.A.) was used as a molecular weight marker. Filters were prehybridized in a solution containing 6 x SSPE [Standard saline phosphate (EDTA)] (1.08 M NaCl, 6 mM EDTA, and 60 mM sodium phosphate, pH 8.3), 2.5 X Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), 50% formamide, and 200 bg/ml of tRNA with the addition of 1 x lo7 cpm of pCA 38.3 oligolabeled with [32P]-dCTP (16). The filters were washed twice with 2 x SSC, 0.5% SDS for 5 min at room temperature; once with 1 X SSC (Standard saline citrate), 0.5% SDS for 30 rnin at room temperature; once with 0.5 X SSC, 0.5% SDS for 30 min at room temperature; and finally, once with 0.1 X SSC, 0.5% SDS for 30 rnin at 62°C. Because we observed that the cDNA probe for CA I1 had a tendency to nonspecifically hybridize to 18s and 28s RNA, total RNA from either C l l D or CAK cells (both are mouse fibroblast cell lines) was used as a negative control in each northern blot. These cell lines were selected since it has been established that CA I1 is not produced by rodent fibroblasts (17,M). We also have examined poly(A +)RNA from C11D by northern blot analysis and found it to be negative under the conditions used in these studies.
CARBONIC ANHYDRASE II IN PANCREATIC ADENOCARCINOMA
For slot blot analysis, CA I1 homologous mRNA was analyzed by filter hybridization using the Minifold I1 Slot Blotter System (Schleicher and Schuell, Keene, NH, U.S.A.). We followed the procedure that was recommended by the manufacturer, which was basically a modification of the method described by Kafatos et al. (19). Three concentrations were used for each RNA sample. The final amounts of RNA applied were 0.05, 0.1, and 0.5 pg brought up to 150 p1 with 20 x SSC (3 M NaCl-0.3 M trisodium citrate). Total RNA from CllD cells was used to assess nonspecific retention of the probe. Hybridization was performed using the CA I1 cDNA probe and the hybridization conditions that were described earlier.
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Analysis of CA I1 protein from tissue and cell lines by immunoblotting Pellets of cultured cells and powdered tissues were sonicated in two volumes of 0.05 M Tris Cl, pH 7.4. The cytosolic proteins were then used immediately for immunoblotting experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out essentially as described by Murakami and Sly (7) except that goat anti-rabbit IgG conjugated with alkaline phosphatase was used as the second antibody. Following treatment of the filter with the second antibody and washing, the filter was stained for 2 min in the dark with a reaction mixture containing 50 ml of 0.06 M borate, pH 9.7; 25 mg of B-naphthyl phosphate; 60 mg of Mg2S0,; and 25 mg of Blue RR and then washed with water. Polyclonal rabbit antibodies to human CA I and CA I1 were kindly provided by Drs. R. E. Tashian and P. J. Venta, University of Michigan Medical School. In some experiments, the proteins were subjected to isoelectric focusing (IEF) gel electrophoresis prior to immunoblotting. In these experiments, agarose IEF gels were made by adding 0.6 g of IEF-agarose (Pharmacia) and 7.2 g of sorbitol to 60 ml of water, heating the solution to 95°C to dissolve the agarose, cooling to 70"C, adding 3.8 ml of ampholine (Pharmalyte, pH 3-10), and casting the gel at 55°C. After minor evaporation, final concentrations were approximately 0.9% agarose, 11% sorbitol, and 5.7% (voVvol) ampholine. We used 1 M NaOH and 0.01 M H2S04 as the cathode and anode solutions, respectively. Gels were prefocused at a 15-mA, 500V, 5-W limit for 30 min, and after loading the samples, focusing was carried out for 2 h with a 15-mA, l,OOO-V, 10-W limit. Immunoblottingwas done pas-
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sively by overlaying the agarose gel with nitrocellulose for 1 h. Subsequent procedures were the same as those in the SDS-PAGE immunoblotting. RESULTS CA I1 homologous RNA in human tissues and pancreatic adenocarcinoma cell lines Poly(Af) RNA was isolated from normal human pancreas, normal human liver, and the following cell lines: MDAPanc-3, Capan-2, MiaPaCa-2, BxPC-3, and PANC-1. Twenty micrograms of each RNA were subjected to blot analysis. As a negative control, 20 pg of total RNA from CAK cells was also run. The filters were hybridized to 32P-labeled pCA 38.3 as described earlier. Following autoradiography, we observed a band at the 1.7-Kb position in all preparations except PANC-1 and the negative control (Fig. 1). The results are also summarized in Table 1. We classified autoradiographic signals detected at that position for MiaPaCa-2 as +- since they approached the limits of detection, with the intensity of the autoradiographic signals ranging between zero and a faint shadow from one run to another. Bands of approximately 4.4 Kb were detected in MDAPanc-3, Capan-2, MiaPaCa-2, BxPC3, and in some cases in the pancreatic RNA, but not in PANC-I, the liver, or CAK. Variability in the intensity of this 4.4-Kb band was observed in the hybridization from one preparation to another.
FIG. 1. Blot analysis of poly ( A + ) RNA from normal human pancreas, liver, and pancreatic adenocarcinoma cell lines. Poly(A+) RNA (20 pg) from the normal human pancreas, normal human liver, and the cell lines Capan-2, MDAPanc-3, Mia PaCa-2, BxPC-3, and PANC-1, as well as total RNA from CAK cells, was fractionated by electrophoresis on agarose gels in 6% formaldehyde and transferred to nitrocellulose paper. Filters were hybridized overnight with 32P-pHCA38.3 to 42T, washed, and subjected to autoradiography as described in Methods and Materials. The positions of the 0.25-9.5-Kb molecular weight markers in the RNA ladder are indicated at the right. Pancreas, Vol. 5 , No. 5 , 1990
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TABLE 1. Species of CA" II homologous poly(A +) RNA detected in tissues and cell lines ~~
~
Cells/tissues
1.7-Kb RNA
4.4-Kb RNA
Pancreas Liver CAK cells MiaPaCa-2 PANC-1 MDAPanc-3 Capan-2 BxPC-3
+
k
a
+-
*+ + +
-
++ + +
CA, carbon anhydrase.
This variability as well as the nature of this band will be discussed in greater detail in the Discussion. CA I1 immunoreactive protein in the pancreas, liver, hemolysates, and pancreatic adenocarcinoma cell lines Cytosolic protein extracts of normal human pancreas and liver and the cell lines PANC-1, MDAPanc-3, BxPC-3, and Capan-2, as well as red blood cell hemolysates, were subjected to SDS-PAGE and then blotted onto nitrocellulose. Normal human liver was utilized as a positive control for CA I1 since pCA 38.3 (the CA I1 cDNA clone used in these studies) was isolated from a cDNA library derived from human liver mRNA. Hemolysates were used as a second positive control since they are highly enriched in both CA I1 and CA I. The filters were treated with polyclonal rabbit antisera to human CA 11, incubated with a goat antibody prepared against rabbit IgG that had been conju-
gated with alkaline phosphatase, and stained as described in Methods and Materials. The results are shown in Fig. 2. We observed a 30-kD band corresponding to CA I1 in each of the samples examined, with the exception of PANC-1. The 30-kD band in BxPC-3 was barely visible. In addition, a second band was observed in the liver, pancreas, and all cell line extracts; it corresponded to a molecular mass of approximately 62 kD. The level of the 62kD band vaned in the tissues from one preparation to another but remained fairly consistent from one preparation to another in the extracts of the different cell lines. The size of this 62 kD band does not correspond to that of any of the previously described members of the CA family. Because the 62kD band falls in a size range that is similar to that of alkaline phosphatase, we considered the possibility that the 62 kD protein might actually be alkaline phosphatase that was reacting with the staining reagents that are designed to detect alkaline phosphatase that is conjugated to the second antibody. This was not the case, because when the CA I1 antibody was omitted from the processing of the filter and the blots were only treated with the second antibody and the staining reagents, or the staining reagents alone, we did not detect the 62-kD band (data not shown). In addition, we carried out a number of experiments in which blots were incubated with the rabbit anti-CA I antisera; the 62-kD band was absent in all cell lines and tissues tested (data not shown). When extracts from pancreatic adenocarcinoma
FIG. 2. Immunoblotting of extracts of normal human pancreas, liver, pancreatic adenocarcinorna cell lines, and hemolysates with carbon anhydrase 11-specificantibody following SDS-PAGE.Extracts from the normal human pancreas, liver, hemolysates, and the cell lines BxPC-3, Capan-2, MDAPanc-3, and PANC-1 were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, electroblottedto nitrocellulosepaper, and incubated with rabbit antisera to CA 11. The filters were then incubatedwith a second antibody to rabbit IgG conjugated with alkaline phosphatase and stained as described in Methods and Materials. Pancreas, Vol. 5 , No. 5 , 1990
CARBONIC ANHYDRASE II IN PANCREATIC ADENOCARCINOMA
tified members of the CA family. The presence of the 62-kD CA I1 immunoreactive protein could occur as a result of any of a number of processes, including posttranslational processing of the 30-kD CA I1 protein (e.g., glycosylation); alternative splice sites in the processing of the CA 11 RNA, resulting in a larger protein product; formation of a stable dimer between two CA I1 molecules; formation of a complex between CA I1 and another protein; biosynthesis of a protein that is the product of a CA 11-related but distinct gene; or biosynthesis of a protein that is unrelated to the CA I1 gene family and fortuitously cross-reacts with the CA I1 antibody. It is not yet known if this band corresponds to the major band that focuses at a more acidic pH in the IEF gels in these same samples. We are analyzing this issue further. A 4.4-Kb RNA was detected in all cell lines except PANC-I and some samples of pancreatic RNA. The nature of this band is not known. That this band was not detected in all pancreas autopsy samples we have examined thus far could reflect a variation in the pancreatic content of this mRNA from one individual to another. For example, it could fluctuate in abundance in response to the nutritional status of the individual from whom the autopsy specimen was obtained. It could also fluctuate because some RNA preparations were more degraded than others. While we expect some variation in the level of degradation from one RNA preparation to another, we do not feel that the degree of variation in the ratios of 18s and 28s RNA (as ascertained by citric acid gel electrophoresis) was sufficient to explain the absence of the 4.4-Kb band in some preparations. Another possibility is that the 4.4-Kb mRNA is only partially homologous to CA 11, and slight variations in the stringency of the hybridization or the washing conditions could result in variability in intensity. It is not likely that either the 62-kD protein or the acidic protein detected on IEF are coded for by the 4.4-Kb RNA, since PANC-1 does not express the 4.4-Kb RNA but does express the two proteins. It is interesting that while CA I1 was not detected in PANC-1 by either immunoblotting or northern blotting, CA activity was detected by Madden and Sarras (21) in this cell line. They assayed homogenates of whole human pancreas and PANC-1 cells using the method of Lonergan and Sargent (22) and found similar levels of CA in each preparation. One explanation for these findings is that the duct cells of the pancreas express more than one member of
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the CA gene family and that while significant levels of CA I1 may not be produced by PANC- 1, levels of the “other” CA enzyme(s) may be relatively high. In conclusion, we detected CA 11 homologous mRNA and CA I1 immunoreactivity with a molecular mass of 30 kD in the pancreas and some cell lines established from pancreatic adenocarcinoma. While the CA I1 immunoreactivity cofocused with CA I1 immunoreactivity in red blood cell hemolysates in IEF studies, we cannot exclude the possibility that the CA 11-like protein and mRNA that we detected are products of a gene that is related to but distinct from the CA I1 gene and do not correspond to any of the genes for previously identified CA family members. Also, we cannot eliminate the possibility that the CA I1 gene products are processed differently in the pancreas, resulting in the production of a CA I1 gene product that is distinct from that in other tissues. In this case, the change in the CA I1 gene product would have to be one that did not alter the isoelectric point. Final proof that the CA I1 gene is expressed in the pancreatic tissue rests ultimately upon the isolation of a cDNA clone prepared from RNA isolated from the human pancreas and proof that the nucleotide sequence of that clone corresponds to that of the established nucleotide sequence for the human liver CA I1 mRNA reported by Montgomery et al. (12) and human kidney CA I1 mRNA reported by Murakami et al. (23). The cloning and nucleotide sequence analysis of the pancreatic CA I1 homologous RNA is planned. Acknowledgment: We thank Terry K. Bertin for his help in developing the immunoblotting techniques. This work was supported in part by a grant from the National Cancer Institute (R01-CA46687) awarded to M.L.F., a grant from the Business and Professional Women of Texas awarded to M.L.F., and Biomedical Research Support grants awarded to M.L.F. (RR-05511) and D.H.E.(RR-07148).
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