P-Selectin Expression in a Metastatic Pancreatic Tumor Cell Line (SUIT-2)'

[CANCERRESEARCH57. 1206-1212. March 5. 997] P-selectin Expression in a Metastatic Pancreatic Tumor Cell Line (SUIT-2)'

Takeshi Iwamura, Thomas C. Caifrey, Norio Kitamura, Hideo Yamanan, Toshiaki Setoguchi, and Michael A. Hoffingsworth2

The Eppley institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68/98-6805 fT. 1., 7'. C. C., M. A. H.], and Surgery 1, Miyazaki Medical College. Miyazaki. Japan fT. 1.. N. K.. H. 1'., T. S.]

ABSTRACT E-selectin is not expressed constitutively but is induced on the surface of endothelial cells upon activation by inflammatory cytokines (inter The human pancreatic tumor cell line SUIT.2 was derived from a leukin 1 or tumor necrosis factor; Refs. 9 and 10). L selectin (Leu 8 metastatic lesion in the liver of a patient with pancreatic adenocarcinoma. and TQ 1) is expressed constitutively at the cell surface and mediates SUIT-2 and clonal cell lines derived from it show spontaneous metastasis to lung and regional lymph nodes from s.c. nude mouse xenografts and the specific homing of lymphocytes into murine peripheral lymph were found to express P-selectin mRNA and protein. Surface expression of nodes as well as the binding of polymorphonuclear neutrophils to P-selectin protein was increased by exposure of the pancreatic tumor cells endothelium in inflamed tissues (11, 12). P-selectin (PADGEM pro to thrombin, oxygen radicals, and trypsin, suggesting that common cellu tein, GMP-140, and CD-62) is located in a granules of resting lar mechanisms for regulating P-selectin surface expression exist among platelets and Weibel-Palade body membranes of endothelial cells (13, platelets, endothelial cells, and these pancreatic tumor cells. The finding 14). Upon activation of endothelial cells and platelets by thrombin that P-selectin is expressed by metastatic pancreatic tumor cells demon (13), histamine (14, 15), or other agents (15â€”18),P-selectin is rapidly strates that the range of cell types that express these adhesion molecules is redistributed to the plasma membrane, where it mediates adhesion of broader than believed previously. activated platelets to monocytes and neutrophils and adhesion be tween endothelial cells and neutrophils (15, 19â€”22).P-selectin is INTRODUCTION believed to play an important role in leukocyte extravasation during cellular inflammatory responses (18, 23). The human pancreatic tumor cell line SUIT-2, which was derived It was hypothesized previously that selectins play a role in tumor from a patient' s liver metastasis, is unusual among human tumor cell metastasis by contributing to adhesion of migrating tumor cells to lines in that it shows spontaneous metastasis to lung and regional endothelial cells (24, 25), based on the fact that many tumor cells lymph nodes from s.c. nude mouse xenografts (1â€”3).There is one express on their cell surfaces high levels of ligands for the selectins other published report of such spontaneous metastatic activity among such as the sialyl Lewis X, sialyl Lewis A, and related carbohydrate human pancreatic tumor cell lines, the FG cell line, which was structures (26â€”32).This report is the first to demonstrate expression established by several cycles of injection of COLO 357 cells into the of P-selectin by human pancreatic adenocarcinoma tumor cells. In spleens and isolation of spontaneous metaStases from the livers of addition, it is shown that cell surface expression of P-selectin is nude mice (4). Twenty-eight clonal sublines of SUIT-2 were derived increased on SUIT-2 cells following treatment with thrombin, oxygen by in vitro methods (5). Two sublines, respectively, were established radicals, and trypsin. by sequential cycles of transplanting SUIT-2 cells to s.c. or i.v. (tail vein) sites in nude mice, harvesting metastases from the lungs, and then repeating this selection process. These clonal cell lines have MATERIALS AND METHODS distinct metastatic and histological properties (2, 3, 5). Six cell lines that show distinct morphological and metastatic properties, ranging Cells and RNA Preparation. A humanpancreatictumorcell line (SUIT from poorly differentiated to well differentiated and from rarely 2), which was derived from a liver metastasis, shows spontaneous metastasis to lung and regional lymph nodes after s.c. injection of tumor cells into the metastatic to highly metastatic, were analyzed by differential display lateral flank of nude mice. Twenty-eight sublines were cloned from SUIT-2 by to identify genes that are differentially expressed among them. culture in soft agar and characterized for their morphological and metastatic One gene differentially expressed by some of the metastatic pan properties (1â€”3).Four sublines were selected for further study (5): 52-007, creatic tumor cell lines is P-selectin. Selectins are expressed on the moderately differentiated tubular adenocarcinoma, highly metastatic; S2-020, surface of endothelia, leukocytes, and platelets and are known to poorly differentiated tubular adenocarcinoma, moderately metastatic; 52-013, mediate initial adhesion interactions between these different cell types well differentiated tubular adenocarcinoma, moderately metastatic; and 52- (6). Selectins have a unique and characteristic structure that includes 028, papillo-tubular adenocarcinoma, rarely metastatic. Two additional mete an extracellular NH2-terminal domain similar to Ca2@-dependent C- static cell lines were established by sequential selection of lung colonies in type lectin, a single epidermal growth factor-like domain, a variable nude mice following tail vein injection (S2-VP1O, moderately differential number of short consensus repeat units similar to domains found in tubular adenocarcinoma, highly metastatic) or spontaneous metastasis to the complement-binding proteins, a transmembrane seg.ient, and a short lung following s.c. injection (S2-CP9, moderately differentiated tubular ade nocarcinoma, very highly metastatic). S2-VP1O and S2-CP9 are cell lines cytoplasmic tail (6, 7, 8). Three members of the selectin family established after 10 and 9 cycles of selection, respectively. All of these cell are currently known. E-selectin (ELAM-1) mediates binding of neu lines have distinct metastatic and morphological properties. They are main trophils and memory T cells to inflamed vascular endothelium. tamed in DMEM supplemented with 5% FBS3. ECV3O4, a cell line derived from spontaneously transformed human umbilical endothelial cells, was ob Received 3/I 8/96; accepted I/I 4/97. tamed from American Type Culture Collection (Rockville, MD) and main The costs of publication of this article were defrayed in part by the payment of page tamed in a 1:1 mixture of DMEM and Ham's F-12 (DMEMIF12) with 5% charges. This article must therefore be hereby marked advertisement in accordance with FBS. One human pancreatic tumor cell line, HPAC, was obtained from Dr. 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported in part by Grants CA 57362, DK 44762, and CA 36727 William Gower (University of South Florida) and another, HPAF, was oh from the NIH, Grant 510 16 from the American Cancer Society, and a grant from the mined from Dr. Richard Metzgar, Duke University Medical Center. The human Nebraska Department of Health. pancreatic tumor cell lines COLO 357, Capan-l, and ASPC-l; human colon 2 To whom requests for reprints should be addressed, at The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68 198-6805. Phone: (402) 559-8343; Fax: (402) 559-4651; 3 The abbreviations used are: FBS, fetal bovine serum; GAPDH, glyceraldehyde-3- E-mail: [email protected]. phosphate dehydrogenase. 1206

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1997 American Association for Cancer Research. P-SELECTIN EXPRESSION IN PANCREATIC CANCER cancer cell lines LS 180, HT-29, and SW480; human breast tumor cell lines were applied for 30 mm at room temperature, respectively, with four washes SK-BR-3, BT-20, and MCF7; human gastric tumor cell line MS; human in PBS between each incubation. Diaminobenzidine (Sigma Chemical Co., ST. duodenal tumor cell line HuTu 80; human malignant melanoma cell line Louis, MO) was used for color development. SK-MEL-28 were obtained from American Type Culture Collection. The lung Western Blotting. The indicated cells were scraped from 225 cm2 flasks cancer cell line, HAIT-l, was established in our laboratory. HPAF, HPAC, and lysed in PBS(â€”) with a Potter-type homogenizer; nuclei were removed by LS18O, HuTu 80, and HAIT-l were grown in DMEMIF12 with 5% FBS. centrifugation at 2000 X g, and protein was quantified by Bradford assay COLO357, Capan-1,ASPC-l, HT-29,SW480, SK-BR-3,BT-20, MCF7,MS. (Bio-Rad, Hercules, CA) prior to Western blotting. Approximately 500 @igof and SK-MEL-28 were grown in DMEM with 5% PBS. protein were used for each cell extract. Approximately 15 p@gof a similar RNA was isolated by using the acid guanidinium thiocyanate-phenol extract from out-of-date platelets were used as a positive control for detecting chloroform extraction method (33). Cells at 80â€”90%confluencewere dis P-selectin in these experiments. Western blotting was performed using tech solved in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, niques reported previously (36). In summary, SDS-PAGE was performed with 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol), followed by the addition of a 3% stacking gel and a 5% running gel; molecular weight markers were the sodium acetate (pH 4.0) to 0.2 M. The solution was extracted with phenol Multimark multi-colored standards (Novex); the samples were electrophoresed (saturated 100 mrsiTris, pH 6.0), chloroform (1:0.1:1:0.2), and RNA was for 600 V-h under reducing conditions and were then transferred to a polyvi precipitated by adding 2.5 volumes ethanol, washed in 75% ethanol, and nylidene difluoride membrane (Millipore) by electroblotting for 350 V-h at redissolved in diethylpyrocarbonate-treated water at 4 @g/ml. 5Â°C.Thepolyvinylidene difluoride membranes were blocked with 10% evap Differential Display. The differential display procedure used here was orated milk in PBS at 5Â°Cfor 48 h. Purified rabbit anti-human CD62P modified from the original method described by Liang and Pardee (34) and (P-selectin) polyclonal antibody (1:500; PharMingen) was incubated with the Liang et al. (35). Anchored oligonucleotide dT primers T12CTwere synthe membranes for 1 h at 25Â°C.The primary antibody was removed, and the sized by the Molecular Biology Core Facility of The Eppley Institute for membrane was washed four times with an excess of PBS(â€”).Following a 1-h Research in Cancer and Allied Diseases. Arbitrary 10-residue oligonucleotides incubation with goat anti-rabbit IgG (H+L) horseradish peroxidase-labeled were purchased from Operon Biotechnology, Inc. (Alameda, CA). In separate antibody (1:1000; Southern Biotechnology, Birmingham, AL), the membrane reactions, 1.0 and 4.0 @goftotal RNA from the indicated cell lines were was washed with PBS(â€”) four times, incubated for 8 mm with SuperSignal reverse transcribed in a reaction containing 1 @MT12C'F,10 mM LifT, 0.5 CL-horseradish peroxidase enhanced chemiluminescent substrate (Pierce), and umt/Ml RNase inhibitor, 200 @Mdeoxynucleotide triphosphates, 1 unitlpi exposed to Enhanced-ECL film (Amersham Corp., Arlington Heights, IL) for Stratascript reverse transcriptase (Stratagene, La Jolla, CA), 1X RT buffer 1â€”60mm. (Stratagene) in a 40-pi reaction volume. One @ilofeach reaction was used for Induction ofSurface Expression ofP-selectin. 52-013, 52-020, ECV3O4, PCR in 1 @tMT12CT,1 @LMOPA-04(AATCGGGCTG), 1.5 mr@iMgC12,100 and MCF7 cell lines were used for studies of induction of P-selectin mRNA or @LMdeoxynucleotide triphosphates, 0.025 pi4d a[32P]dATP, 1 X PCR buffer, surface expression of P-selectin protein. Five thousand cells in 100 j.@lof and 0.125 unitlpi Taq DNA polymerase in 10 @lreaction volume. The cycling DMEM/F12 with 5% PBS were placed in 96-well microtiter plates (Dynatech parameters were: 4 cycles of 94Â°C,30 s; 40Â°C,2 mm; 72Â°C,2 mm; then 36 Laboratories, Inc., Chantilly, VA). After 4 days of culture, at 90â€”100%cell cycles of 94Â°C,30s; 44Â°C,30s; and 72Â°C,1mm. Taq DNA polymerase was confluence, all of the cell lines were exposed to 0.5 unit/ml of human thrombin purchased from Life Technologies, Inc. (Grand Island, NY). a[32P]dATP was (Sigma) dissolved with DMEMIF12with 5% PBS, washed twice with DMEM, purchased from Amersham International (Buckinghamshire, England). The then incubated with 100 ,d of DMEMIF12 with 5% PBS. At times prior to amplified cDNAs were separated on 5.0 and 3.0% denatured polyacrylamide thrombin exposure and 10, 20, 30, 45, 60, and 90 mm, and 2, 3, 4, 5, 6, 8, 12, gels at 2000 V for 3 h, and radioactivity was quantified and visualized on a and 24 h after treatment with thrombin, the cells were washed with PBS(â€”) Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). twice, then fixed with 2% paraformaldehyde in PBS(â€”). In separate experi Cloning of Differential Display Products and DNA Sequencing. Differ ments, 52-013 cells were exposed to thrombin for 20 mm or continuously for entially displayed cDNA fragments were recovered from denaturing polyacryl 24 h and then fixed as described above or used to purify total RNA. In other amide gels, reamplified, gel-purified as described above, ligated into the PCR experiments, 52-020, ECV3O4, and MCF7 were exposed to thrombin for 10 II vector, and cloned by using the TA cloning kit (Invitrogen, San Diego, CA). latin and evaluated in a similar manner until 8 h after exposure to thrombin. In cDNA inserts were isolated following agarose gel electrophoresis by centrif another set of experiments,the indicated cells were treated for 1 or 10mm with ugation on Millipore Ultrafree-MC (0.22 p.m filter unit; Milipore, Bedford, thrombin, 1 mm with 0.25% trypsin in PBS(-), or 1 h with 250 mM of hydrogen MA). peroxide in DMEM/F12 with 5% PBS. The cells were fixed as described above cDNA fragments that gave evidence of differential mRNA expression by at the indicated time points up to 3 h after treatment. Northern blot analysis were sequenced in both directions (Sequenase kit Fixed cells were washed twice with PBS(â€”),200,d of 5% skim milk were version 2.0; U. S. Biochemical, Cleveland, OH) by using oligonucleotide added to each well, and the plates were incubated at room temperature for 3 h. primers for M13 reverse and 11. Sequences of cDNA fragments were corn After discarding the milk, 50 @lofanti-P-selectinantibody solution [0.5 mg/nil pared to known sequences by searching the GenBank date base with the in PBS(â€”)] were added to each well. Plates were incubated at 37Â°Cfor 2 h and FASTA program (Genetics Computer Group, Madison, WI). washed gently with PBS(â€”) with 0.05% Tween 20. Ten thousand cpm of Northern Blots. A cDNA for P-selectin (bp 383-1110) obtained from the WI-labeled goat antimouse immunoglobulin (Cappel, Durham, NC) in differential display reaction was used to probe Northern blots. A cDNA insert PBS(â€”)wereadded to each well, and plates were incubated at room temper for GAPDH was used as a positive control for RNA quantity and quality. ature for 1 h. Following three times washes with PBS(â€”),boundantibody was The probes were radiolabeled by using the Rediprirne DNA labeling system quantified in a gamma counter (Clinigamma; LKB-Wallac, Turku, Finland). (Amersham) and used as probes in Northern blot analysis. Northern blots were Cells cultured on a chamber slide (as described above) were treated with prepared with 20 i@gof total RNA from each of the indicated cell lines. thrombin (0.5 unitlml, in DMEM/Fl2 with 5% PBS) for 20 mm, washed twice Immunohistochemlcal Staining. 52-013, S2-020, ECV3O4, HPAC, and with DMEM, then incubated in 200 pA of DMEMIFI2 with 5% PBS. Before HPAF cells were cultured on chamber slides (Lab-Tek Chamber slide; Nunc, exposure and 30 and 60 mm and 2, 3, 4, and 6 h after exposure to thrombin, Inc., Naperville, IL), washed three times with Ca2@-and Mg@k-free PBS the cells were washed twice with PBS(â€”),fixedwith 2% paraformaldehyde in [PBS(â€”)],andfixed with2%paraformaldehydeinPBS(â€”)for1h. Fixedcells PBS(â€”),andevaluated for P-selectin expression by using the immunoperoxi were evaluated for P-selectin protein expression by using P-selectin-specific dase method described above. and -negative control antibodies and the streptavidin-biotin-horseradish per oxidase staining kit (Histomark Streptavidin-HRP system; Kirkegaard & Perry Laboratory, Inc., Gaithersburg, MD). Briefly, internal peroxide was blocked RESULTS with 0.3% H202 in methanol. Nonspecific antibody binding was blocked with normal goat serum for 30 rain at room temperature. Cells were covered with Isolation of a Differentially Expressed cDNA by Differential I mg/mi ofanti-P-selectin monoclonal antibody (clone AC1.2, IgGl, sc;Becton Display. Differential display was used to identify genes differentially Dickinson Cellular Imaging Systems, San Jose, CA) for 1 h at room temper expressed among six clonal cell lines derived from the human pan ature. The secondary biotinylated antibody and then streptavidin-peroxidase creatic adenocarcinoma cell line SUIT-2 that show distinct properties 1207

P-SELECTIN EXPRESSION IN PANCREATIC CANCER

Immunohistochemical Staining. Expression of P-selectin protein in SUIT-2 and cell lines derived from it was investigated by using an immunoperoxidase technique with antibody AC.1 (Becton Dikinson), which is specific for P-selectin. Consistent with the results of the @ @O4B1@@,@, ,s@ Northern blotting experiments, the S2-013 cell line showed expression @@@@ (P-eelectln) :@ @, @.#4!W s,@ *s@ of P-selectin protein (Fig. 3), whereas no expression was detected in ECV3O4, PANC-1, and MCF-7 (data not shown). Antibody reactivity at the perimeter of the cells is consistent with the interpretation that P-selectin is expressed at high levels on the cell surface of S2-0l3 cells (Fig. 3B). Western Blotting. The molecular properties of P-selectin detected in cell lines derived from SUIT-2 was investigated by Western blotting. @ /@5@/ C/C/Cf P-selectin expressed by S2-0l3 cells showed the same mobility in SDS 25ng of total RNAused In RT PCR bOng of total RNAused In RT PCR PAGE as P-selectin expressed by platelets, with an apparent molecular

Fig. 1. Results of differential display performed on six sublines derived from the mass of approximately 148,000 Da (Fig. 4). P-selectin was not detected SUrr-2 cell line. Twenty-five and 100 ng of total RNA from each cell line were reverse in lysates of the HT29 colon adenocarcinoma cell line (Fig. 4). transcribed using the T12C'I' primer and then PCR amplified with T12C1' primer and arbitrary OPAO4 primer. The arrow shows the band designated C1'O4Bl, which was Induction of Surface Expression of P-selectin. Cell surface ex differentially amplified in the 52-007 and 52-013 cells. pression of P-selectin on S2-013 cells before and after treatment with thrombin, which is known to induce surface expression on platelets and endothelial cells, was quantified by RIA. Cell surface expression of of morphology (cellular differentiation) and metastasis. The results of P-selectin on S2-013 cells was increased following 20 mist ofexposure to one set of differential display reactions that used primers T12CT and thrombin, and maximal levels were attained between 30 and 60 ruin (Fig. OPA-04 are shown in Fig. 1. One cDNA fragment (CTO4BJ, arrow) 5). At periods between 1 and 3 h after treatment, levels of P-selectin was detected only in the cell lines S2-007 and S2-0l3. This cDNA decreased almost 2-fold compared to levels detected prior to thrombin fragment was recovered, reamplified, and cloned using the pCR H exposure (Fig. 5). Thereafter, cell surface P-selectin increased during the vector. An insert of approximately 700 bp was obtained, which was period from 3 to 12 h after thrombin exposure to levels not significantly similar in size to that seen in the differential display reaction. different from the pretreatment steady-state levels (Fig. 5). Continuous Northern Blot Hybridization. Fig. 2 shows the results of probing exposure to thrombin for 24 h did not result in a significant superinduc a Northern blot of RNA from several tumor cell lines and the SUIT-2 tion of P-selectin expression above that seen with 20 miii exposure cell lines with the CTO4B 1 cDNA fragment. SUIT-2, S2-007, and (Fig. 5). S2-0l3 show high levels of expression, and S2-028 shows very low Northern blot experiments were used to determine if treatment of levels of expression of this mRNA (difficult to see in the figure). S2-013 cells with thrombin affected the steady-state levels of P Three sublines derived from SUIT-2 (S2-020, S2-CP8, and S2-VP1O) selectin mRNA. The results of these experiments (Fig. 6) show that and 19 human cell lines from pancreatic, colon, and lung adenocar steady-state levels of P-selectin mRNA by S2â€”013 cells undergo cinomas do not show expression of this mRNA. subtle but reproducible fluctuations following treatment with throm Sequencing of cDNA Fragments. Sequence analysis of the bin. There is a 2-fold decrease in steady-state P-selectin mRNA by CTO4B1 cDNA in both directions revealed that both the 3' and 5' 180 mm, followed by an increase to levels 50% higher than steady ends of CTO4B1 cDNA fragment contained the OPA-04 primer se state levels by 360 mm, and a return to steady-state levels by 480 mm. quence. The cloned insert showed greater than 98% identity in se Thus, fluctuations in steady-state mRNA are a mirror image (inverse) quence to the cDNA for P-selectin including bp 383-1110 [GenBank; of fluctuations in the levels of surface expression of P-selectin fol release 91.0 (10/95)]. lowing treatment with thrombin (Fig. 5); the alterations in mRNA

Origin â€”

Fig. 2. Northem blot analysis of P-selectin mRNA expression in tumor cell lines. SUIT-2. 52- 28$ 007, and S2-0l3 show high levels of expression, and 52-028 shows very low levels of expression of P-selectin P-selectin mRNA. Three sublines derived from (3.2 kbp) SUIT-2 (52-020, S2-CP8, and S2-VP1O) and 17 human tumor cell lines from pancreatic (PANC-l, HPAC, HPAF, COLO 357, Capan- 1, and AsPC- 1), las @.- colon (HT-29, LS 180, and SW480), breast (5K- BR-3, BT-20, and MCF7), duodenal (HuTu8O), gastric (MS), and lung (HArr-l) adenocarcinomas, @@ malignant melanoma (SK-MEL-28), and a sponta GAPDH a @. neously transformed endothelial cell line (ECV3O4) do not show expression of P-selectin mRNA.

@c:,rl@c@c:,r@ . c@%L@_@_Ll:@ - @-

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Fig. 3. Immunohistochemical staining of 52-013 cells with a monoclonal antibody against P-selectin. A, negative control monoclonal antibody of the same isotype as ACI.2. B. monoclonal antibody AC1.2 shows reactivity in the cytoplasm and at the perimeter of the cells, consistent with the interpretation that high levels of P-selectin are found on the cell surface of 52-013 cells.

levels are similar in magnitude and duration, and they occur at times 1 h later than fluctuations in levels of expression of surface protein. @ I4SkD Thrombin treatment did not induce detectable surface expression of (P@se1ectin) p P-selectin on three tumor cell lines (S2-020, ECV3O4, and MCF7; Fig. 7). Surface expression of P-selectin by S2-013 cells was induced to levels similar to those seen following thrombin exposure by treatment

. a with oxygen radicals or trypsin; likewise, a similar pattern of increase, . decrease, and recovery was observed (Fig. 8). The results of immu nohistochemical analysis were also consistent with the finding of induction of surface expression of P-selectin by these agents (data not shown).

DISCUSSION

Previous examination of the normal tissue distribution of P selectin showed that it is expressed only on the surface of activated platelets and endothelial cells and not on other normal epithelial of the pancreas or other organs (14). It has been reported previously Fig. 4. Western blot demonstrating P-selectin expression by platelets and pancreatic adenocarcinoma cell lines. The pancreatic tumor cell lines 52-013 and 52-020 express that P-selectin is found on 37% of primary breast cancers examined P-selectin that is similar in molecular weight to P-selectin that is expressed by platelets, by immunohistochemical techniques (37, 38), although it was not as detected by probing a Western blot with a polyclonal anti-P-selectin antibody. P conclusively established that the tumors produced P-selectin. The selectinis not expressedbythe colontumorcell line HT29.Resultswithcontroland preimmune rabbit antisera were negative for reactivity with proteins of similar molecular data presented in this report show unequivocally that P-selectin weight (data not shown). mRNA and protein is constitutively expressed by the human pan creatic tumor cell line SUIT-2, and that modulation of surface expression of P-selectin on these pancreatic tumor cells is regu

(cpm) â€”0â€” : n=5, mean Â±s.d., 20 mm exposure to thrombin

500 .â€”.â€” : n=5, mean Â± s.d., continuous exposure to thrombin gnlll : negative control ( background binding value, meanÂ± s.d.) Fig. 5. Induction of surface expression of P-selec tin in S2-0l3 cells following treatment with throm bin. MA of P-selectin expression on S2-013 cells following treatment with thrombin (0.5 unit/mI) for 20 mm. Time was measured after exposure to throm bin. Surface expression of P-selectin on S2-013 in creased rapidly following treatment with thrombin; 300 maximal levels were attained between 30 and 60 rein after thrombin treatment. At periods between 120and 180 mm, surface expression of P-selectin decreased to belowthoselevelsseenpriorto thrombinexpo- 200 sure. During the period up to 720 miss after thrombin exposure, cell surface expression of P-selectin in creased to levels not significantly different from pre

@ treatment steady-state levels. Bars, SD. U 0 120 240 360 480 600 720 840 960 1080 1200 1320 1440 Time after exposure to thrombin (mm)

1209

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1997 American Association for Cancer Research. P-SELECTIN EXPRESSION IN PANCREATIC CANCER (x1O-@) 8 Fig. 6. Quantification of P-selectin mRNA expression by 52-013 cells following exposure to thrombin. 52-013 7 * cells were exposed to thrombin (0.5 unit/mI) for 20 rein I and then used to quantify mRNA levels for P-selectin. a. 6 f :n@ Time was measured after exposure to thrombin. The * : P < 0.05 intensity of a P-selectin probe on Northern blots (similar 5 in quality to those shown in Fig. 2) was quantified on a Fujix bioimaging analyzer BAS1000-MacBAS (Fuji Film 4 Co.,Tokyo,Japan).ThesameNorthernblotwasstripped and rehybridized with a GAPDH probe. Quantified values 3 ofsignal strength for the P-selectin probe were divided by values obtained for the GAPDH probe. Mean values (bars, SD) of the P-selectin:GAPDH ratio for three mdc 2 pendent experiments are presented. Statistical signifi cance was determined by Student's t test. 0 120 240 360 480 600 720 840 960 1080 1200 1320 1440 Time after exposure to thrombin (mm) lated in a manner that is similar to that reported previously for proposed that interactions between the cell surface adhesion mol activated platelets and endothelial cells. Several other human tu ecules on tumors and endothelium is important to the process of mor cell lines, including six pancreatic adenocarcinomas, three metastasis and angiogenesis. These hypotheses and findings lead colon adenocarcinomas, three breast adenocarcinomas, a human us to propose that expression of P-selectin contributes to two gastric carcinoma, a duodenal carcinoma, a lung adenocarcinoma, aspects of the biology and clinical course of pancreatic adenocar and a melanoma, did not express P-selectin mRNA. cinoma: metastasis and thrombosis. P-selectin has been shown to play an important role in cell Regarding metastasis, it has been hypothesized that tumor cells use adhesion events in the vasculature during episodes of acute inflam selectin-mediated cellular adhesion interactions as a means of traf mation (19, 20, 39â€”42). Surface expression of P-selectin is rapidly ficking through the body during the process of metastatic spread of induced upon activation of platelets and endothelial cells. P-selec malignant cells. Evidence in support of this hypothesis includes the tin expressed on the surface of these cells has been shown to fact that many adenocarcinomas (including pancreatic) express on facilitate adherence of neutrophils and monocytes to platelets and their cell surfaces carbohydrate structures such as sialyl Lewis X, endothelial cells. In addition, increased expression of adhesion sialyl Lewis A, and others that are known to function as selectin molecules including P-selectin has been observed in areas of ligands when expressed on appropriate protein backbones (26, 32, tumors undergoing neovascularization (37, 38), and it has been 42â€”44).The cell line evaluated in this study, SUIT-2, expresses sialyl

n=5, mean Â±s.d.,10 mm exposure to thrombin (cpm)

S2-020

0 180 360 540 720 Time after exposure to thrombin (mm)

Fig. 7. Induction of surface expression of P-selectin in S2-020, (cpm) ECV3O4,and MCF7 cells following thrombin (0.5 unit./ml)expo sure for 10 mm. RIA of P-selectin expression on the indicated cell lines following treatment with thrombin (0.5 unit/ml) for 10 mm. ECV3O4 Time was measured after exposure to thrombin. Surface expression of P-selectin was not induced on S2-020, ECV3O4, and MCF7 cells. Bars, SD. 0 180 TImeafter exposure to thrombin (mm)

(cpm)

MCF-7

180 360 540 Time after exposure to thrombin (mln) 1210

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1997 American Association for Cancer Research. P-SELECTIN EXPRESSION IN PANCREATIC CANCER f :n.'5,meanÂ±s.d. I_ :negativecorttrol(backgroundbindingvalue,meanÂ±s.d.)

Thrombmn(1mm) Thrombun(10mm) H202(1h) Trypsmn(1mm) (cpm) (cpm@ (cpm) 1500 1500

1250 1250

1000 1000

750 750

I .Jw 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 TImeafterexposure -rimeafterexposure â€¢rlmeafterexposure TIme after exposure to thrombin(mm) tothrombin(mm) to H2O@(mm) to trypsmn(mm)

Fig. 8. Induction of surface expression of P-selectin in 52-013 following treatment with thrombin, H2O2, or trypsin. RIA of P-selectin expression on 52-013 cells treated with: thrombin(0.5unit/mi)for1and10mm;H2O2(250ms) for 1h;ortrypsin[0.25%inPBS(â€”)]forI mm.Timewasmeasuredafterexposuretotheindicatedagents.Surfaceexpression of P-selectin by 52-013 cells was induced rapidly by treatment with oxygen radicals or trypsin, and a pattern of increase, decrease, and recovery similar to that seen with thrombin treatment was observed. Bars, SD.

Lewis A (1). The expression of both P-selectin and its ligand by selectin protein by S2-013 cells is increased by exposure of the SUIT-2 and clonal lines derived from it raises the possibility that these cells to thrombin, oxygen radicals, and trypsin suggests that com molecules play a role in adhesion properties between individual mon cellular mechanisms exist among platelets, endothelial cells, tumor cells. and these pancreatic tumor cells for regulating surface expression One characteristic that distinguishes SUIT-2 from the other of P-selectin. This supports the hypothesis that P-selectin cx pancreatic tumor cell lines evaluated here is that SUIT-2 shows pressed by tumors is responsive to natural factors that regulate its spontaneous metastatic activity when xenografted s.c. into nude biological activity. Patients with pancreatic tumors frequently mice (1â€”3).Constitutive expression of P-selectin may contribute to present with thrombotic lesions during the course of their disease the unusual spontaneous metastatic activity of this cell line. Con (47), and the patient from which SUIT-2 was derived had evidence sistent with this hypothesis are two findings: three clonal cell lines of disseminated thromboses (l).@ This leads us to propose the derived from SUIT-2, which show moderate to well-differentiated hypothesis that expression of P-selectin by adenocarcinoma cells morphology (S2â€”007 and S2â€”013) and spontaneous metastatic contributes in part to the formation of thromboses in patients with activity to lung and lymph nodes, express P-selection; and one pancreatic adenocarcinoma. clonal cell line that is well differentiated and rarely metastatic In summary, the SUIT-2 pancreatic adenocarcinoma cell line cx (S2â€”028) expresses very low levels of P-selectin (Fig. 2). There is presses P-selectin and is responsive to natural factors (thrombin, not an absolute correlation between the metastatic properties of all cell lines derived from SUIT-2 and P-selectin expression; however, oxygen radicals, and trypsin) that modulate the surface expression of because two cell lines produced by several cycles of in vivo this adhesion molecule. Additional studies should be performed to selection for lung colonization (by spontaneous metastasis, S2- establish the incidence of P-selectin expression by adenocarcinomas CP9, or from tail vein injections, S2-VP1O) did not express P and to determine if its expression contributes to the development of selectin (Fig. 2). metastatic and thrombotic lesions. P-selectin expression on the surface of platelets and other cells plays a significant role in their aggregation and adherence during episodes of coagulation and inflammation. P-selectin is not con ACKNOWLEDGMENTS stitutively expressed on the plasma membrane of endothelial cells We thank Angie Rizzino for helpful suggestions. and platelets; it is stored in granules. Surface expression is induced by external stimuli, such as exposure to thrombin (13, 45, 46) or oxygen radicals (16). The finding that surface expression of P 4 T. Iwamura, personal observation. 1211

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Takeshi Iwamura, Thomas C. Caffrey, Norio Kitamura, et al.

Cancer Res 1997;57:1206-1212.

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