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[Cancer Research 66, 898-906, January 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Identification of a Putative Tumor Suppressor Gene Rap1GAP in Pancreatic Cancer

Lizhi Zhang1, Li Chenwei1, Redah Mahmood1, Kenneth van Golen2, Joel Greenson3, Gangyong Li1, Nisha J. D'Silva4, Xiangquan Li2, Charles F. Burant2,5, Craig D. Logsdon6,7 and Diane M. Simeone1,5

Departments of 1 Surgery, 2 Internal Medicine, 3 Pathology, 4 Oral Medicine, and 5 Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan; Departments of 6 Cancer Biology and 7 Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Diane M. Simeone, University of Michigan Medical Center, TC 2922D, Box 0331, 1500 East Medical Center Drive, Ann Arbor, MI 48109. Phone: 734-615-1600; Fax: 734-936-5830; E-mail: simeone{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly down-regulated in pancreatic cancer. Only a small percentage of preneoplastic pancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer. (Cancer Res 2006; 66(2): 898-906)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pancreatic cancer is a formidable disease with a 5-year survival rate <5%. A major hallmark of pancreatic cancer is its extensive local tumor invasion and early systemic dissemination. These features likely contribute to the fact that the vast majority of patients present with advanced disease and cannot be offered curative treatment. The molecular basis for these characteristics of pancreatic cancer is incompletely understood. A better understanding of the genes involved in tumor growth and invasion may allow development of novel treatment strategies to tackle this rapidly progressive disease.

We recently found that Rap1 GTPase-activating protein 1 (rap1GAP) was significantly down-regulated in pancreatic cancer (1). Rap1GAP, a 663-amino-acid protein with a molecular weight of 73 kDa, was the first identified member of a family of proteins with GAP function specific for Rap1 (2). Rap1 belongs to the Ras family of small GTP-binding proteins and is implicated in a host of cellular processes, including proliferation, adhesion, and morphogenesis (3–5). Activation of Rap1, like other Ras family proteins, occurs by cycling between GTP-bound and GDP-bound forms. The kinetics of Rap1 GTP hydrolysis and GDP dissociation are regulated by two protein classes: guanine-nucleotide exchange factors (GEF) and GAPs. GEFs promote the release of bound GDP and the association with a new GTP molecule, which activates Rap1. GAPs stimulate the low intrinsic GTPase activity of Rap1 and induce GTP hydrolysis, thus inactivating Rap1 in cells.

Rap1GAP is a member of a family of related GAP proteins, each with a unique expression profile and cellular distribution (6). Little is known about the role of rap1GAP in cancer, although alterations in other rap1GAP family members play a role in cancer development and/or progression. SPA-1 is a rap1GAP expressed in peripheral T cells and hematopoietic progenitors in bone marrow. Mice deficient in SPA-1 developed a spectrum of myeloid disorders that resemble chronic myeloid leukemia (7). Another member of the rap1GAP family, E6TP1, is targeted for ubiquitin-mediated degradation by the viral E6 oncoprotein and its virally mediated down-regulation may contribute to cervical cancer and other cancers associated with chronic human papillomaviruses infection (8). Tuberin, the product of the tuberous sclerosis type 2 (TSC2) gene, is a rap1GAP homologue and functions as a GAP for the Ras-like protein Rheb (9). Mutations in the GAP domain of tuberin cause tuberous sclerosis (10) and somatic inactivation of TSC2 predisposes to formation of sporadic gliomas (11).

In this study, we examined the role of rap1GAP in pancreatic tumor growth and progression. We verified that rap1GAP is down-regulated in pancreatic cancer and provide data to support its critical role in pancreatic cancer progression. Furthermore, we also show that a significant number of pancreatic cancer patients have loss of heterozygosity (LOH) of the rap1GAP gene located at chromosome 1p36.1-p35, indicating that rap1GAP may be a novel tumor suppressor gene in pancreatic cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Pancreatic tissue and pancreatic cancer cell lines. Paraffin-embedded human pancreatic tissues came from the University of Michigan Medical Center and conformed to the policies and practices of the University of Michigan Institutional Review Board. The pancreatic cancer cell lines MIA PaCa-2, Panc-1, BxPC-3, CAPAN-2, HPAF-11, HPAC, AsPc-1, Su 8686, and CFPAC were obtained from the American Type Culture Collection (Manassas, VA). The immortalized pancreatic ductal cell line HPDE was kindly provided by M.S. Tsao (University of Toronto, Toronto, ON, Canada).

Immunohistochemical analysis. A pancreatic cancer tissue microarray containing five samples of normal pancreas and 47 samples of pancreatic adenocarcinoma was used for immunohistochemical analysis of rap1GAP expression. Immunohistochemistry was done using a standard biotin-avidin complex technique (Vector Labs, Burlingame, CA) and a rabbit polyclonal antibody against rap1GAP (Santa Cruz, Santa Cruz, CA) at a dilution of 1:1,000. Twenty-five paraffin-embedded human pancreatic tissue samples containing pancreatic intraductal neoplasia (PanIN) lesions of varying stages were also stained with an anti-rap1GAP antibody. Rap1GAP expression in PanIn lesions was evaluated by an experienced pancreatic pathologist and graded as negative, weak positive or positive.

Creation of stable cell lines. Cell lines with stable expression of rap1GAP were generated by transfecting Panc1 and MiaPaCa-2 cells with a pcDNA3.1 vector containing Flag-rap1GAP. Control cell lines were generated by transfecting Panc-1 and MiaPaCa-2 cells with an empty pcDNA3.1 vector. Selection for neomycin resistance was initiated 48 hours after transfection by adding 500 µg/mL of G418 (Life Technologies) to the culture medium. The selection medium was changed every 3 days for 5 weeks. Clones of G418-resistant cells were isolated and expanded for further characterization.

Reverse transcription-PCR. Reverse transcription-PCR (RT-PCR) was done using total RNA prepared from human samples of normal pancreas, chronic pancreatitis and pancreatic cancer. Reverse transcription was conducted for 45 minutes at 45°C from 500 ng of purified RNA in 25 µL of Reserve Transcription system reaction mixture using avian myeloblastosis virus reverse transcripase (Promega, Madison, WI). Reverse transcription was followed by 25 cycles of PCR (30 seconds denaturation at 94°C, 30 seconds annealing at 58°C, and 30 seconds extension at 72°C). The primers designed for rap1GAP were as follows: forward 5'-GGAGCTTCGCGCGCCCAACAACC-3' and reverse, 5'-CTCCGTCCACACCCTCCGTCTCCT-3'. Primers designed for ribosomal protein S6 (RPS6) were as described (12). Amplified products were separated on 1.5% agarose gels and visualized by ethidium bromide. Images of the RT-PCR ethidium bromide–stained agarose gels were acquired with a Gel Doc EQ system and quantified using Multi-Analyst Version1.1 software (Bio-Rad Laboratories, Hercules, CA). Rap1GAP mRNA expression was normalized to RPS6 expression levels from the same samples.

Immunoblot analysis. Immunoblot analysis was done as previously described (1) using rabbit polyclonal anti-phospho-focal adhesion kinase (Upstate, Lake Placid, NY), rabbit polyclonal anti-total focal adhesion kinase and anti-Rap1, goat polyclonal anti-rap1GAP (Santa Cruz), and mouse anti-Flag antibodies (Sigma, St. Louis, MO). Images were visualized using the ECL Detection System (Amersham, Arlington Heights, IN). Film images were scanned with an Agfa Arcus II (Bayer Corp., Ridgefield Park, NJ) to create a digital image.

Rap1 activity assays. Rap1 activity assays were done according to instructions of the manufacturer (Upstate). Briefly, whole cell lysates were prepared by incubating the cells in ice-cold lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 0.5 mol/L NaCl, 1% NP40, 2.5 mmol/L MgCl2, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10% glycerol]. The cells were sonicated for 5 seconds and the lysates centrifuged at 14,000 x g for 5 minutes at 4°C. The supernatant was removed and assayed for protein concentration using the Bio-Rad protein assay. Thirty microliters of Ral GDS-RBD agarose were added to each tube containing 0.5 mL cell extract and then incubated for 45 minutes at 4°C. Pellet beads were collected by centrifugation (10 seconds, 14,000 x g) and washed thrice with lysis buffer. The beads were resuspended in 40 µL of 2x Laemmli buffer and 2 µL of 1 mol/L DTT was added, followed by boiling for 5 minutes. Western blotting was done to detect Rap1.

Cell proliferation assays. Cell proliferation was measured using a CellTiter 96 AQueous nonradioactive cell proliferation assay (Promega) as we have previously described (13).

Soft-agar colony formation assays. Assays of colony formation in soft agar were done by preparing 1 mL underlayers consisting of 0.6% agar medium in 35 mm dishes by combining equal volumes of 1.2% Noble agar (Difco, Detroit, MI) with 2x DMEM and 20% fetal bovine serum (FBS). Cells were trypsinized, centrifuged, and resuspended in 0.3% agar medium followed by plating onto the previously prepared underlayers. The cells were kept wet by adding a small amount of DMEM with 10% FBS every other day. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere for 4 weeks and were then stained with methylene blue. Stained colonies were photographed and counted.

Detection of apoptosis by flow cytometry. To detect apoptotic cells, an ApoAlert Annexin V-FITC Apoptosis kit was used (BD Biosciences, Palo Alto, CA). Cells were induced to undergo apoptosis with serum starvation and treatment with 5-fluorouracil (5-FU, 300 µg/mL) or etoposide (200 µg/mL) for 16 hours. Cells were collected and rinsed once with binding buffer. Resuspended cells were stained with Annexin V-FITC/propidium iodide according to the instructions of the manufacturer. Flow cytometry using a single laser emitting excitation light at 488 nm was done to detect Annexin V–positive cells.

Cell motility assays. Motility was assessed using colloidal gold random motility assays as described (14). Motility was induced by stimulating the cells with 10% FBS. Phagokinetic tracks were visualized by microscopy and were digitally recorded. The areas of the phagokinetic tracks were measured using ImageJ version 1.30 software and expressed as pixels squared.

Immunofluorescent staining of cytoskeleton and focal adhesion components. Cells were serum starved for 24 hours and then were stimulated with 10% FBS for 45 minutes. Immunofluorescent staining for phalloidin and vinculin was done to detect focal adhesion and F-actin assembly using a FAK100 kit from Chemicon International, Inc. (Temecula, CA). Briefly, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then were permeabilized with 0.1% Triton X-100 for 5 minutes. After blocking with 1% bovine serum albumin for 30 minutes, the cells were incubated with a monoclonal antivinculin antibody for 1 hour at room temperature. After washing, the slides were incubated with FITC-conjugated secondary antibody and tetramethyl rhodamine isothiocyanate (TRITC)–conjugated phalloidin antibody for 30 minutes. Nuclei counterstaining was done by incubating cells with 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes. The slides were mounted using a Vectashield mounting solution (Vector Labs). Fluorescent images were visualized and recorded with an Olympus FV-500 confocal microscope.

In vitro cell invasion assays. Matrigel invasion assays were done as previously described using precoated Transwell filters with 8 µm pores (14).

Orthotopic pancreatic cancer model. Male CB17 severe combined immunodeficient (SCID) mice (Charles River, Wilmington, MA) were housed under pathogen-free conditions in accordance with University of Michigan Animal Care and Use Committee guidelines. Ten-week-old mice were anesthetized with an i.p. injection of xylazine (9 mg/kg) and ketamine (100 mg/kg). A median laparotomy was done and an aliquot of 5 x 105 MiaPaCa-2 cells (empty vector or rap1GAP stably transfected) in a volume of 30 µL was injected into the pancreatic tail using a 30-gauge needle (six per group). To prevent leak from the injection site, the needle was slowly withdrawn after a 1-minute delay and slight pressure was applied with a sterile cotton swab. The mice were sacrificed 7 weeks later and autopsies were done. Primary tumor size was measured and tumor volume was calculated using the formula for hemi-ellipsoids: V = length (mm) x width (mm) x height (mm) x 0.5236 (15). The extent of local invasion and distant metastasis was also recorded. Primary and metastatic lesions were excised, fixed in 10% formalin, and embedded in paraffin. Tissue sections were obtained and stained with H&E.

LOH analysis of rap1GAP. DNA was extracted from nine microdissected samples of human pancreatic cancer, nine pancreatic cancer cells lines, and the immortalized human pancreatic ductal cell line C6H11. A rap1GAP–specific marker, rap1GA1 3+4 (GDB:1006983), was selected from the Genome Database (www.gdb.org). This marker is contained within intron 9 of the rap1GAP gene at chromosome 1p36.1-p35. Primer sequences to amplify this region were designed from the Genome Database and PCR primers were purchased from Invitrogen (Carlsbad, CA). Forty nanograms of DNA were used as a template in each sample. PCR was done in a 25 µL reaction volume containing 0.2 µmol/L PCR primers, 1x PCR Master Mixer (Promega), 1x PCR buffer, 200 µmol/L deoxynucleotide triphosphate, 1.5 mmol/L MgCl2, and 0.6 units of Taq DNA Polymerase. DNA was amplified for 30 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, followed by a 10-minute extension at 72°C. PCR products were evaluated using a DNA 500 Labchip kit and analyzed using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). We used the formula of Cawkwell et al. (16) with a change in allelic ratios of 0% to 50% scored as LOH. This criterion permits LOH detection in tumor samples with up to 50% stromal contamination, as is seen with pancreatic adenocarcinoma.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rap1GAP1 is down-regulated in pancreatic cancer. Because significant evidence has emerged that dysregulation of Rap1 activation is responsible for the development of malignancy in a variety of cell types (17), we determined if alterations in the Rap1 signaling pathway might contribute to pancreatic tumor development and/or progression by examining expression levels of a variety of components of the Rap1 signaling pathway. According to our gene profiling data (1), expression levels of Rap1{alpha} and Rap1ß, both isoforms of Rap1, were not altered in pancreatic cancer. The expression levels of Rap1 GEFs, which can activate Rap1, including C3G and Epacs, were also unchanged in pancreatic cancer. Analysis of rap1GAP family members revealed that rap1GAP levels were significantly down-regulated in pancreatic cancer, whereas other rap1GAP family members, including TSC2 and SPA-1, were not significantly changed.

Rap1GAP was significantly down-regulated in pancreatic cancer and pancreatic cancer cell lines, compared with both normal and chronic pancreatitis samples (Fig. 1A). Rap1GAP expression was lower in chronic pancreatitis than normal pancreas, likely because the large number of stroma cells in chronic pancreatitis do not express rap1GAP by immunohistochemistry (data not shown). We used RT-PCR to independently examine mRNA levels for rap1GAP in five samples each of normal pancreas and pancreatic adenocarcinoma and four samples of chronic pancreatitis. RT-PCR showed expression of rap1GAP in normal pancreas and chronic pancreatitis but little or no expression of rap1GAP in pancreatic cancer samples (Fig. 1B). RPS6 served as an internal control.


Figure 1
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  		Figure 1. Down-regulation of rap1GAP in pancreatic cancer correlates with pancreatic cancer progression status. A, cDNA microarray analysis (1) was done using Affymetrix chips containing 6,800 genes. Microdissected samples from pancreatic adenocarcinoma (10), pancreatic cancer cell lines (7), chronic pancreatitis (CP, 5), and normal pancreas (5) were analyzed. mRNA expression levels of rap1GAP were expressed as Affymetrix units. *, P < 0.05. B, validation of microarray results of rap1GAP mRNA levels using RT-PCR analysis. Total RNA was collected from pancreatic adenocarcinoma (5), chronic pancreatitis (4), and normal pancreas (5), and 100 ng was subjected to RT-PCR for rap1GAP mRNA. RPS6 mRNA was used as an internal control for variance in loading samples. Representative of a single experiment. Experiments were repeated twice with essentially the same results. C, summarized results from analysis of the level of rap1GAP expression in individual samples in the pancreatic cancer tissue array containing five samples of normal pancreas and 47 samples of pancreatic adenocarcinoma. Columns, percentage of total sample type. D, samples from the tissue array were graded as well (8), moderate (25), and poorly differentiated (14) and the extent of rap1GAP expression was quantitated as negative (white columns), weakly positive (gray columns), or positive (black columns). Columns, percentage of total sample type. E, rap1GAP immunostaining of two samples of pancreatic adenocarcinoma. High-power photomicrograph of rap1GAP immunostaining demonstrating strong staining of benign pancreatic ducts on the bottom and negative staining of a well-differentiated pancreatic adenocarcinoma on the top (left). High-power photomicrograph of rap1GAP immunostaining showing weak staining of pancreatic acinar cells on the right and negative staining of poorly differentiated pancreatic adenocarcinoma cells on the left (right).

 
Using a pancreatic cancer tissue microarray, we next evaluated the expression of rap1GAP protein in samples of normal pancreas (n = 5) and pancreatic adenocarcinoma (n = 47). The expression level of rap1GAP in individual samples was scored as negative, weak positive, and positive by a blinded pancreatic pathologist. Rap1GAP protein expression was observed primarily in the cytoplasm (Fig. 1E). In normal tissue, strong staining of rap1GAP was observed in ductal and islet cells with weak staining in acinar cells. In rap1GAP-expressing pancreatic cancers, staining was seen only in the neoplastic epithelium. Most pancreatic cancer samples were graded as negative (28 of 47, 60%), whereas 40% were weakly positive. These results are summarized in Fig. 1C and confirm a significant down-regulation of rap1GAP levels in pancreatic adenocarcinoma.

To determine if the expression of rap1GAP correlated with the differentiation status of pancreatic adenocarcinoma, cancer samples on the tissue microarray were graded as well, moderately, and poorly differentiated. The level of rap1GAP expression was then analyzed in a blinded manner and correlated with tumor differentiation status. We found a direct correlation between loss of rap1GAP expression with more poorly differentiated tumors. Over 65% (9 of 14) of poorly differentiated cancers lost rap1GAP expression compared with 25% (2 of 8) of well-differentiated cancers (Fig. 1D). We did not observe a correlation between rap1GAP expression levels and patient survival (data not shown); however, it is likely that a larger study group would be needed for adequate statistical power to determine the effect of rap1GAP expression on this end point.

Rap1GAP expression in PanIN lesions. A progression model of pancreatic cancer has become widely accepted in which normal duct epithelium progresses to infiltrating cancer through a series of morphologically defined pancreatic cancer precursors named PanINs (18). PanIN1 lesions show hyperplasia without dysplasia, PanIN2 lesions show dysplasia, and PanIN3 lesions show severe nuclear atypia characteristic of carcinoma in situ. This progression is associated with the accumulation of specific genetic alterations, such as K-ras mutation and inactivation of p16 and p53. To examine if loss of rap1GAP expression is associated with pancreatic tumorigenesis, we measured rap1GAP expression in 25 human pancreatic tissue samples (19 cancers and 6 chronic pancreatitis) containing PanIN lesions. Only a small proportion of PanIN lesions lost rap1GAP expression, with 2 of 12 (17%) PanIN1, 2 of 13 (15%) PanIN2, and 2 of 11 (18%) PanIN3 lesions showing loss of rap1GAP expression, with no evidence of progressive loss of rap1GAP expression with PanIn progression. This contrasts to the observed 60% loss of rap1GAP expression in invasive pancreatic cancers (Fig. 1C). These results suggest that inactivation of rap1GAP is not critical in the early stages of pancreatic cancer development but rather occurs at a later stage of tumor progression.

Overexpression of rap1GAP blocks Rap1 activation in pancreatic cancer cells. To examine the functional role of rap1GAP in pancreatic cancer, we increased rap1GAP expression in pancreatic cancer cells by stably transfecting the human pancreatic cancer Panc1 and MiaPaCa-2 cells with a Flag-tagged rap1GAP vector or empty expression vector as a control. Results obtained from the two different cell lines were similar and, therefore, experiments described in this report are from the MiaPaca-2 cell line. Two independent clonal cell lines of Flag-rap1GAP-transfected MiaPaCa-2 cells (designated R1 and R2) showed increased rap1GAP expression compared with the corresponding wild-type and control vector-transfected cells (Fig. 2A). High basal Rap1 activity was detected in nontransfected and control empty vector-transfected cancer cells. However, in rap1GAP-overexpressing cells (R1 and R2), Rap1 activity was completely abolished (Fig. 2B). Thus, introduction of rap1GAP into pancreatic cancer cells results in inhibition of Rap1 binding activity.


Figure 2
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  		Figure 2. Inhibition of cancer cell proliferation and survival by rap1GAP. A, a representative immunoblot demonstrating levels of rap1GAP expression in MiaPaCa-2 cells stably expressing rap1GAP and their wild-type and vector control transfectants (top). The same membrane was stripped and reblotted with anti-Flag antibody showing overexpression of the transfected Flag-tagged rap1GAP gene (bottom). B, Rap1 activation is inhibited in rap1GAP stably transfected cancer cells. Rap1 activity assay was done in MiaPaCa-2 cells. Thirty microliters of Ral GDS-RBD agarose were used in 0.5 mL cell extract to pull down the activated form of Rap1 (GTP-Rap1). Western blotting was done to detect the GTP-Rap1 (top). The same amount cell extract (30 µg) was also used to perform a regular Western blotting with anti-Rap1 antibody as an internal control for loading (bottom). Similar results were also observed in Panc-1 cells. Representative of a single experiment. Experiments were repeated twice with essentially the same results. C, the R1 and R2 rap1GAP-transfected MiaPaCa-2 cell lines and vector-transfected control cells were plated in 96-well plates at a density of 500 cells per well in 150 µL DMEM with 5% FBS for 5 days. At each day, 15 µL/well of combined 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate solution was added and the absorbance was determined by using an ELISA plate reader. Points, average absorbance of three wells in one experiment. The experiment was repeated twice with similar results. D and E, colony formation in soft agar. Five thousand parent cells or their transfectants per well were plated in 35 mm dishes containing 0.6% agar medium (underlay) and 0.3% agar medium (uplayer). The cells were incubated at 37°C in a humidified 5% CO2 atmosphere for 4 weeks and were then stained with methylene blue. Stained colonies were photographed and counted. A representative stained well for control and rap1GAP-transfected cells is shown in Fig. 4B. The summary results from three separate experiments are presented in Fig. 4C. *, P < 0.01. F, rap1GAP promotes cell apoptosis. Apoptosis was induced by treating rap1GAP-transfected MiaPaCa-2 cells or controls with either 5-FU (300 µg/mL) or etoposide (200 µg/mL) for 16 hours. Cells were collected and stained with Annexin V-FITC/propidium iodide. Flow cytometry was done to detect Annexin V–positive cells. Experiments were repeated thrice. *, P < 0.01.

 

Figure 4
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  		Figure 4. Rap1GAP may function by regulating integrin activity. A, confocal fluorescence microscopy of focal adhesion and F-actin formation in MiaPaCa-2 cells. Focal adhesion formation was detected using antivinculin monoclonal antibody and a FITC-conjugated secondary antibody (green), and F-actin was detected using TRITC-conjugated phalloidin (red). Nuclei were counterstained using DAPI (purple). B, Western blot analysis of the effect of rap1GAP expression on phosphorylation of focal adhesion kinase (p-FAK) following serum exposure for indicated times. The same membrane was stripped and reblotted with anti–total focal adhesion kinase antibody to show equal loading. The experiment was repeated thrice with similar results.

 
Overexpression of rap1GAP inhibits cell proliferation and survival. Rap1 has been shown to be important in regulation of cell proliferation; however, its effects are cell type specific. For example, overexpression of Rap1 in Swiss 3T3 cells induces DNA synthesis and decreases doubling time (3, 19), but introduction of constitutively activated Rap1 in astrocytes (20) and in keratinocytes (21) inhibits cell proliferation. To examine whether increasing rap1GAP expression influences the growth properties of pancreatic cancer cells, we examined the effect of rap1GAP on in vitro proliferation rates and the ability of cells to form colonies in soft agar. Rap1GAP-transfected R1 and R2 cell lines showed a significant decrease in proliferation (Fig. 2C). The effect of rap1GAP was even more pronounced in inhibiting anchorage-independent growth, measured by colony formation (Fig. 2D and E).

Resistance to apoptosis is a key factor in the survival of malignant cells. To determine the role of rap1GAP in pancreatic cancer cell apoptosis, we examined the effect of rap1GAP on basal apoptotic rates and in response to the apoptosis-inducing chemotherapeutic drugs 5-FU and etoposide using Annexin V staining. The basal apoptotic rate of rap1GAP-overexpressing pancreatic cancer cells increased 2-fold compared with control vector-transfected cells (data not shown). Treatment with 5-FU or etoposide for 16 hours resulted in an apoptosis rate of 4% to 5% in control MiaPaCa-2 cells; however, rap1GAP-overexpressing cells showed much higher rates of apoptosis in response to 5-FU and etoposide (28% and 13%, respectively; Fig. 2F). These results suggest that loss of rap1GAP expression in human pancreatic adenocarcinoma may render cells resistant to apoptosis and, therefore, play a role not only in tumor progression but chemotherapeutic resistance.

Rap1GAP inhibits cell motility and invasion ability in vitro. Increasing cell motility and invasion are two key components of tumor progression. Several studies have documented that Rap1 functions in integrin-mediated morphologic changes, cell adhesion, and cell motility (22). To examine whether rap1GAP expression is associated with cancer cell invasiveness, we measured motility and invasion in parental MiaPaCa-2 cells and rap1GAP transfectants using colloidal gold random motility and in vitro invasion assays. As seen in Fig. 3A, R1 and R2 cells showed a significant decrease in random motility, measured by white cell movement tracks, compared with control pancreatic cancer cells, which showed wider and longer white tracks. Quantitation of track area revealed a 68% (R1) and 60% (R2) decrease in cell motility compared with control cells (Fig. 3B). In vitro invasive assays showed that the invasion ability of R1 and R2 cells was decreased 60% compared with wild-type and control vector-transfected MiaPaCa-2 cells (Fig. 3C). These results show that rap1GAP expression is inversely associated with motility and invasiveness of pancreatic cancer cells in vitro.


Figure 3
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  		Figure 3. Effect of rap1GAP on cell motility and invasion. A and B, random cell motility assays were done using a colloidal gold random motility assay. A, representative pictures for control and the rap1GAP-transfected cell lines R1 and R2. B, summary results from three separate experiments. C, in vitro invasion assays were done by determining the percentage of rap1GAP-transfected and control-transfected cells that penetrated through Matrigel-coated Transwell chambers. Experiments were repeated thrice. *, P < 0.05.

 
Rap1GAP may function through integrins. Rap1 has been reported to affect cell function through effects on the Ras/extracellular signal-regulated kinase (ERK) signaling pathway and activation of integrins. We observed no difference in phospho-ERK or total ERK levels between control pancreatic cancer cells and their rap1GAP-transfected counterparts (data not shown). We next determined the effect of rap1GAP on two integrin-dependent events, focal adhesion formation, and F-actin formation. Control MiaPaCa-2 cells stimulated with serum (Fig. 4A, left) manifested morphologic changes suggestive of integrin activation, such as spreading and nuclear enlargement. F-actin formation, assessed by TRITC-conjugated phalloidin staining, was observed at the leading edge of spreading cells. Vinculin, a component of focal adhesion protein complexes, translocated from the cytoplasm to the cell membrane at the leading edge following serum exposure (Fig. 4A, arrows). In contrast, MiaPaCa-2 cells overexpressing rap1GAP did not show morphologic changes associated with integrin activation after 45 minutes of exposure to serum (Fig. 4A, right).

The effect of rap1GAP on integrin activity was further examined by testing the ability of rap1GAP to inhibit focal adhesion kinase phosphorylation. Control MiaPaCa-2 cells showed focal adhesion kinase activation as early as 5 minutes after 2.5% serum treatment, with peak stimulation at 20 to 40 minutes, with return toward control values by 60 minutes (Fig. 4B, top). Focal adhesion kinase activation was not detected in cells overexpressing rap1GAP (Fig. 4B, bottom). These data further support the role of rap1GAP in modulating focal adhesion formation and integrin activation in pancreatic cancer cells.

Rap1GAP inhibits tumor formation and progression in vivo. To determine whether rap1GAP can suppress tumorigenesis and metastasis in vivo, we used an orthotopic SCID mouse model. Mice were injected with 0.5 x 106 vector-transfected or rap1GAP-expressing MiaPaCa2 cells into the pancreatic tail. Animals were sacrificed 7 weeks after injection and pathologic examination was done. All six animals injected with control MiaPaCa-2 cells developed large pancreatic tumors with extensive invasion to local structures, including the portal vein, stomach, and spleen (Fig. 5A and B). Control mice also had extensive liver and peritoneal metastasis (Fig. 5C). However, the mice injected with rap1GAP stably transfected MiaPaCa-2 cells had significantly smaller primary tumors (76.4 ± 25.2 mm3 of R1 versus 320.4 ± 98.7 mm3 of control; Fig. 5A and B), with two of six animals demonstrating no gross tumor. Only one of six mice in the rap1GAP animal group had liver and peritoneal metastases, which were much smaller and fewer in number than those seen with the control animals (Fig. 5C). These results show that overexpression of rap1GAP suppresses primary pancreatic cancer growth and metastasis in an orthotopic pancreatic cancer model.


Figure 5
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  		Figure 5. The effect of rap1GAP expression on tumor growth and metastasis in vivo. A, mean primary tumor size in animals injected with 0.5 x 106 rap1GAP-transfected or vector-control transfected MiaPaCa-2 cells into the tail of the pancreas of SCID mice (six mice per group). B, a representative example of a resected primary tumor and liver from a SCID mouse injected with wild-type MiaPaCa-2 cells compared with a resected primary tumor from a mouse injected with rap1GAP-expressing cells. Note the large primary tumor with extensive desmoplastic reaction and liver metastases in the control animal, whereas the rap1GAP-overexpressing tumor is much smaller, with no evidence of local invasion and or liver metastases. C, incidence of grossly evident primary tumor, liver, peritoneal, and lymph node metastases (Mets), and evidence of local invasion in control (gray columns) and rap1GAP-expressing tumors (black columns).

 
High frequency of LOH of rap1GAP gene at chromosome 1p36.1-p35 in pancreatic cancer. Like other cancers, pancreatic adenocarcinoma is thought to arise from a multistep process of accumulation of genetic alterations, including activating mutations of specific oncogenes, such as K-ras, and loss of function of specific tumor suppressor genes, such as p16, p53, and DPC4 (23). To determine the mechanism of rap1GAP expression in pancreatic cancer, we explored whether the epigenetic mechanism of hypermethylation at CpG dinucleotide sites in or near key cis-acting transcriptional regulatory elements was the cause of inactivation of rap1GAP, particularly because a CpG-rich island is located 2.5 kb upstream of the rap1GAP transcription initiation site. Treatment of four pancreatic cancer cell lines with absent rap1GAP expression (MiaPaCa-2, Panc1, BxPC3, and CAPAN) with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine did not induce rap1GAP expression (data not shown). This suggested that hypermethylation may not cause rap1GAP down-regulation in pancreatic cancer.

LOH at loci on chromosome 1p is frequent in pancreatic adenocarcinoma (23, 24) but the gene(s) targeted for inactivation by 1p LOH are not known. Rap1GAP is located at human chromosome 1 p36.1-p35, a region characterized as a peak of LOH frequency in pancreatic cancer. To determine if a mutational mechanism was responsible for rap1GAP inactivation in pancreatic cancer, we examined the LOH status of rap1GAP in pancreatic cancer. We chose a marker within intron 9 of the rap1GAP gene (Rap1GA1 3+4, GDB:1006983) from the Genome Database and designed PCR primers to amplify this region. DNA was isolated from nine primary pancreatic adenocarcinomas using PCR, and the PCR products were analyzed using DNA LabChips. Three of the nine primary tumors analyzed (33.3%) showed LOH, evidenced by losing one PCR product from one of two alleles (Fig. 6A and B). One of nine cancers showed homozygous deletion with this marker. Furthermore, six of nine pancreatic cancer cell lines (MiaPaCa-2, Panc1, BxPC3, CAPAN, HPAF-11, and HPAC) also showed LOH with this marker, whereas AsPc1, Su8686, CFPAC, and the immortalized pancreatic ductal cell line HPDE did not have LOH. Lack of rap1GAP expression in pancreatic cancer cell lines correlated with LOH status (Fig. 6C). The percentage of LOH we observed was similar to a previous report of LOH frequency (41%) at chromosome 1p36.1-p35 in pancreatic cancer (25). These results suggest that rap1GAP is a putative tumor suppressor gene important in the progression of pancreatic cancer.


Figure 6
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  		Figure 6. LOH of rap1GAP gene in pancreatic cancer. A and B, representative pictures show LOH with PCR products from cancer and normal tissue analyzed using an Agilent 2100 Bioanalyzer. A larger allele (Nl) and a smaller allele (Ns) are shown as two peaks in (A). PCR products on a pseudogel are depicted on the right, demonstrating loss of the larger allele (Tl), whereas a smaller allele (Ts) is still present in the tumor sample. C, immunohistochemical analysis of rap1GAP staining in rap1GAP LOH negative and positive cells. Lack of rap1GAP expression correlates with LOH status. A, 293 cells; B, Su8686 cells; C, BxPC-3 cells; D, Panc-1 cells. Red, rap1GAP; green, actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we have identified a putative tumor suppressor function for rap1GAP, a protein that regulates the Rap1 signaling cascade, in pancreatic cancer. Specifically, we have shown that rap1GAP expression is down-regulated in primary pancreatic cancers and pancreatic cancer cell lines compared with normal pancreas and chronic pancreatitis and that loss of rap1GAP expression correlates with poor differentiation status of tumors. Rap1GAP expression is infrequently lost in PanIN lesions, suggesting that loss of rap1GAP expression comes at a later stage in pancreatic cancer progression. Furthermore, using both in vitro and in vivo models, we have shown that increased rap1GAP expression is associated with the decreased growth, motility, and invasive capability of pancreatic cancer cells, presumably through modulating activation of integrins. Finally, we show that there is a high frequency of LOH of rap1GAP gene at human chromosome 1p36.1-p35, suggesting that rap1GAP may function as an important tumor suppressor gene in pancreatic cancer.

Rap1 was first identified as a protein that could revert the morphologic phenotype of Ras-transformed cells, suggesting that Rap1 either mediates growth-inhibitory signals or interferes with Ras-effector signaling (26). However, later studies suggested that Rap1 delivers an oncogenic signal, making it unclear whether Rap1 plays a "good role" or is a "bad actor" in cancer.

Alterations in other molecules that regulate Rap1 signaling have been found to play a role in oncogenesis in a cell type–specific fashion, including E6TP1, SPA-1, and tuberin (7, 8, 11). Mutations in RapGEFs have also been identified in human tumors and cancer cell lines. For example, myeloid leukemias in BXH-2 mice contain a proviral insertion of the CalDAG-GEF1 gene and the leukemia cells exhibited constitutive Rap1 activation (27). These studies suggest that increased Rap1 activity correlates with oncogenesis.

Very little is known about the role of rap1GAP in cancer development. Rap1GAP is expressed in a tissue-specific manner (28) with low levels of expression in proliferating cells and an increase in expression levels with differentiation (6). A recent report by Tsygankova et al. (29) showed that rap1GAP levels are dynamically regulated in thyroid-stimulating hormone–dependent thyroid cells and suggest that rap1GAP may regulate thyroid cell proliferation and differentiation. In a separate report, cDNA microarrays were used to examine follicle stimulating hormone–induced gene expression in ovarian cancer cells compared with immortalized normal human ovarian cells (30). Rap1GAP was one of nine genes induced by follicle stimulating hormone in normal ovarian cells but not in cancer cells, suggesting that rap1GAP might possess tumor suppressor activity. In this study, we have shown that rap1GAP is down-regulated in pancreatic cancer and that rap1GAP likely serves a tumor suppressor function based on our findings that restoration of rap1GAP activity inhibits tumor growth, invasion, and apoptosis in both in vitro and in vivo studies.

An important question is whether the effects of rap1GAP are mediated solely through alterations in Rap1 activity or if rap1GAP elicits signals distinct from negative regulation of Rap1 activity. One of the most consistent findings of Rap1 function is its involvement in integrin-mediated cell adhesion and motility (31, 32). Integrins are heterodimeric intracellular molecules that possess a unique ability to regulate cell adhesion and have been found to be important in the progression and spread of cancer. It has been firmly established that overexpression of active Rap1 activates integrins and that inhibition of Rap1 signaling inhibits integrin function. In the current study, we assessed if the effects of rap1GAP on pancreatic cancer cell function might be mediated through integrin-dependent events (focal adhesion formation and focal adhesion kinase activation). We found that overexpression of rap1GAP in pancreatic cancer cells blocked focal adhesion formation and focal adhesion kinase activation, effects similar to those seen by inhibition of Rap1 signaling in other cell types, supporting the concept that rap1GAP may be functioning solely by altering Rap1 activity levels.

Although not addressed in this study, other studies suggest that rap1GAP may have actions distinct from its effects on Rap1. For example, in thyroid cells, whereas overexpression of rap1GAP and the dominant-negative Rap1A17N both impaired Rap1 activity, they had opposite effects on growth rate and differentiated gene expression (33). In addition, in a yeast two-hybrid screen, rap1GAP interacted with the Gz member of the Gi family of trimeric G proteins independent of the ability of rap1GAP to interact with Rap1 (34), raising the possibility that different multiprotein complexes containing rap1GAP may exist and elicit distinct cellular activities.

To address mechanisms underlying loss of rap1GAP expression in pancreatic cancer, we examined both epigenetic and mutational mechanisms of rap1GAP gene inactivation. Our data indicate a high frequency of LOH of rap1GAP, with loss of expression of rap1GAP in pancreatic cancer likely due loss of the second allele of the gene. The rap1GAP gene is located on chromosome 1p, which has been long suspected, on the basis of LOH and other data, to harbor a tumor suppressor gene important in pancreatic cancer and other tumors. The chromosomal region in which rap1GAP is located, 1p36-p35, has been identified to site of high frequency not only in pancreatic adenocarcinoma (24, 25) but also in other tumors, including neuroblastoma (35, 36), breast cancer (37), hepatocellular carcinoma (38), hepatoblastoma (39), and endocrine neoplasia (40). However, a specific candidate tumor suppressor gene within this region has not been identified. Further studies will be needed to assess the LOH status of rap1GAP in other tumors with a high frequency of LOH at chromosome 1p36-35. Because of the length and complexity of rap1GAP1 gene (27 exons spanning over 100 kb), significant work will be required to analyze the mutational status of rap1GAP in pancreatic cancer.


   Acknowledgments
 
Grant support: NIH grant DK061507 (D.M. Simeone) and Michigan Life Sciences Corridor grant MEDC03-622 (D.M. Simeone and C.D. Logsdon).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Tom Giordano (Department of Pathology, University of Michigan, Ann Arbor, MI) for providing the pancreatic cancer tissue array slides.

Received 8/24/05. Revised 9/26/05. Accepted 10/26/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Logsdon CD, Simeone DM, Binkley C, et al. Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer. Cancer Res 2003;63:2649–57.[Abstract/Free Full Text]
  2. Rubinfeld B, Munemitsu S, Clark R, et al. Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1. Cell 1991;65:1033–42.[CrossRef][Medline]
  3. Altschuler DL, Ribeiro-Neto F. Mitogenic and oncogenic properties of the small G protein Rap1b. Proc Natl Acad Sci U S A 1998;95:7475–9.[Abstract/Free Full Text]
  4. Ozaki N, Shibasaki T, Kashima Y, et al. cAMP-GEFII is a direct target of cAMP in regulated exocytosis. Nat Cell Biol 2000;2:805–11.[CrossRef][Medline]
  5. Asha H, de Ruiter ND, Wang MG, Hariharan IK. The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J 1999;18:605–15.[CrossRef][Medline]
  6. Kurachi H, Wada Y, Tsukamoto N, et al. Human SPA-1 gene product selectively expressed in lymphoid tissues is a specific GTPase-activating protein for Rap1 and Rap2. Segregate expression profiles from a rap1GAP gene product. J Biol Chem 1997;272:28081–8.[Abstract/Free Full Text]
  7. Ishida D, Kometani K, Yang H, et al. Myeloproliferative stem cell disorders by deregulated Rap1 activation in SPA-1-deficient mice. Cancer Cell 2003;4:55–65.[CrossRef][Medline]
  8. Singh L, Gao Q, Kumar A, et al. The high-risk human papillomavirus type 16 E6 counters the GAP function of E6TP1 toward small Rap G proteins. J Virol 2003;77:1614–20.[CrossRef][Medline]
  9. Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, Pan D. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol 2003;5:578–81.[CrossRef][Medline]
  10. Manning BD, Cantley LC. Rheb fills a GAP between TSC and TOR. Trends Biochem Sci 2003;28:573–6.[CrossRef][Medline]
  11. Gutmann DH, Saporito-Irwin S, DeClue JE, Wienecke R, Guha A. Alterations in the rap1 signaling pathway are common in human gliomas. Oncogene 1997;15:1611–6.[CrossRef][Medline]
  12. Binkley CE, Zhang L, Greenson JK, et al. The molecular basis of pancreatic fibrosis: common stromal gene expression in chronic pancreatitis and pancreatic adenocarcinoma. Pancreas 2004;29:254–63.[CrossRef][Medline]
  13. Zhang L, Duan CJ, Binkley C, et al. Simeone DM. A transforming growth factor ß-induced Smad3/Smad4 complex directly activates protein kinase A. Mol Cell Biol 2004;24:2169–80.[Abstract/Free Full Text]
  14. van Golen KL, Wu ZF, Qiao XT, Bao LW, Merajver SD. RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. Cancer Res 2000;60:5832–8.[Abstract/Free Full Text]
  15. Maeda H, Segawa T, Kamoto T, et al. Rapid detection of candidate metastatic foci in the orthotopic inoculation model of androgen-sensitive prostate cancer cells introduced with green fluorescent protein. Prostate 2000;45:335–40.[CrossRef][Medline]
  16. Cawkwell L, Lewis FA, Quirke P. Frequency of allele loss of DCC, p53, RBI, WT1, NF1, NM23 and APC/MCC in colorectal cancer assayed by fluorescent multiplex polymerase chain reaction. Br J Cancer 1994;70:813–8.[Medline]
  17. Hattori M, Minato N. Rap1 GTPase: functions, regulation, and malignancy. J Biochem 2003;134:479–84.[Abstract/Free Full Text]
  18. Hruban RH, Goggins M, Parsons J, Kern SE. Progression model for pancreatic cancer. Clin Cancer Res 2000;6:2969–72.[Free Full Text]
  19. Yoshida Y, Kawata M, Miura Y, et al. Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes. Mol Cell Biol 1992;12:3407–14.[Abstract/Free Full Text]
  20. Apicelli AJ, Uhlmann EJ, Baldwin RL, et al. Role of the Rap1 GTPase in astrocyte growth regulation. Glia 2003;42:225–34.[CrossRef][Medline]
  21. D'Silva NJ, Mitra RS, Zhang Z, et al. Rap1, a small GTP-binding protein is upregulated during arrest of proliferation in human keratinocytes. J Cell Physiol 2003;196:532–40.[CrossRef][Medline]
  22. Bos JL, de Bruyn K, Enserink J, et al. The role of Rap1 in integrin-mediated cell adhesion. Biochem Soc Trans 2003;31:83–6.[Medline]
  23. Bardeesy N, DePinho RA. Pancreatic cancer biology and genetics. Nat Rev Cancer 2002;2:897–909.[CrossRef][Medline]
  24. Hilgers W, Tang DJ, Sugar AY, Shekher MC, Hruban RH, Kern SE. High-resolution deletion mapping of chromosome arm 1p in pancreatic cancer identifies a major consensus region at 1p35. Genes Chromosomes Cancer 1999;24:351–5.[CrossRef][Medline]
  25. Hahn SA, Seymour AB, Hoque AT, et al. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res 1995;55:4670–5.[Abstract/Free Full Text]
  26. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M. A ras-related gene with transformation suppressor activity. Cell 1989;56:77–84.[CrossRef][Medline]
  27. Dupuy AJ, Morgan K, von Lintig FC, et al. Activation of the Rap1 guanine nucleotide exchange gene, CalDAG-GEF I, in BXH-2 murine myeloid leukemia. J Biol Chem 2001;276:11804–11.[Abstract/Free Full Text]
  28. Polakis PG, Rubinfeld B, Evans T, McCormick F. Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells. Proc Natl Acad Sci U S A 1991;88:239–43.[Abstract/Free Full Text]
  29. Tsygankova OM, Feshchenko E, Klein PS, Meinkoth JL. Thyroid-stimulating hormone/cAMP and glycogen synthase kinase 3ß elicit opposing effects on Rap1GAP stability. J Biol Chem 2004;279:5501–7.[Abstract/Free Full Text]
  30. Ho S-M, Lau K-M, Mok SC, Syed V. Profiling follicle stimulating hormone-induced gene expression changes in normal and malignant human ovarian surface epithelial cells. Oncogene 2003;22:4243–56.[CrossRef][Medline]
  31. Reedquist KA, Ross E, Koop EA, et al. The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J Cell Biol 2000;148:1151–8.[Abstract/Free Full Text]
  32. Caron E. Cellular functions of the Rap1 GTP-binding protein: a pattern emerges. J Cell Sci 2003;116:435–40.[Abstract/Free Full Text]
  33. Tsygankova OM, Saavedra A, Rebhun JF, Quilliam LA, Meinkoth JL. Coordinated regulation of Rap1 and thyroid differentiation by cyclic AMP and protein kinase A. Mol Cell Biol 2001;21:1921–9.[Abstract/Free Full Text]
  34. Meng J, Glick JL, Polakis P, Casey PJ. Functional interaction between G{alpha}z and Rap1GAP suggests a novel form of cellular cross-talk. J Biol Chem 1999;274:36663–9.[Abstract/Free Full Text]
  35. Fong CT, Dracopoli NC, White PS, et al Loss of heterozygosity for the short arm of chromosome 1 in human neuroblastomas: correlation with N-myc amplification. Proc Natl Acad Sci U S A 1989;86:3753–7.[Abstract/Free Full Text]
  36. Martinsson T, Sjoberg RM, Hedborg F, Kogner P. Deletion of chromosome 1p loci and microsatellite instability in neuroblastomas analyzed with short-tandem repeat polymorphisms. Cancer Res 1995;55:5681–6.[Abstract/Free Full Text]
  37. Nagai H, Negrini M, Carter SL, et al. Detection and cloning of a common region of loss of heterozygosity at chromosome 1p in breast cancer. Cancer Res 1995;55:1752–7.[Abstract/Free Full Text]
  38. Yeh SH, Chen PJ, Chen HL, Lai MY, Wang CC, Chen DS. Frequent genetic alterations at the distal region of chromosome 1p in human hepatocellular carcinomas. Cancer Res 1994;54:4188–92.[Abstract/Free Full Text]
  39. Kraus JA, Albrecht S, Wiestler OD, von Schweinitz D, Pietsch T. Loss of heterozygosity on chromosome 1 in human hepatoblastoma. Int J Cancer 1996;67:467–71.[CrossRef][Medline]
  40. Williamson C, Pannett AA, Pang JT, et al. Localisation of a gene causing endocrine neoplasia to a 4 cM region on chromosome 1p35-p36. J Med Genet 1997;34:617–9.[Abstract/Free Full Text]



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