p16 Expression Adenovirus Vector to Suppress Pancreas Cancer Cell Proliferation

The prognoses of pancreatic cancer patients have been miserable even after radical surgery, and adjuvant therapy is necessary to improve the surgical results. p16 (p16) is tight-binding and inhibitory protein for cyclin-dependent kinase 4 to induce G1 arrest of the cell cycle. p16 gene deletion is frequently identified in human pancreas cancer. The impaired gene function of p16 might be a major factor of the uncontrolled proliferation and malignancy of pancreas cancer cells. In this study, we investigated the effect of adenovirus p16 expression vector for pancreas cancer cell proliferation to clarify whether the vector might be a promising mode to assist the surgical therapy for pancreas cancer. We constructed the adenovirus p16 expression vector AdexCACSp16 by inserting p16 cDNA to a cassette cosmid containing a nearly full-length adenovirus type 5 genome with E1 and E3 deletions. Thereafter, we assessed the activity of AdexCACSp16 to induce p16 gene mRNA expression in pancreas cancer cell line MIAPaCa-2 and to control cell proliferation. AdexCACSp16 induced a high level of p16 gene mRNA expression in MIAPaCa-2 cells with 1 h contact to the cells. The cell proliferation was significantly suppressed by AdexCACSp16 compared with the control adenovirus group. These data indicate that AdexCACSp16 has the potential to induce p16 gene expression and control pancreas cancer cell proliferation and that the adenovirus p16 expression vector AdexCACSp16 might be a possible method of gene therapy to improve the surgical therapeutic results for pancreas cancer. INTRODUCTION The recurrence of pancreas cancer occurs frequently, even after radical surgery, and the prognosis compared with other organs is extremely poor, despite many clinical trials (1). The overall 5-year survival rate of pancreas cancer is ,20% and is only ;25% among curative cases (2, 3). These data indicate that there is a limitation on surgical treatment for pancreas cancer and that approaches other than past adjuvant methods are necessary to improve the survival rate. The deletion of the p16 (p16) gene is identified frequently in human cancer cell lines. The gene MTS1, which was sought as the deleted locus in human cancer chromosomes, has been found to be identical with this p16 gene (4, 5). It has been reported that homozygous or heterozygous deletion of the p16 gene is identified frequently in the human cancers with poor prognoses such as pancreatic and esophageal cancer. Caldas et al. (6) reported that allelic deletions of the p16 locus were detected in 85% of informative cases and that they found homozygous deletions in 15 (41%) and sequence changes in 14 (38%) among 10 cell lines and 27 xenografts from human pancreas cancer. On the other hand, the p16 deletion is rare in gastric, colon, and hepatocellular carcinomas, whose prognoses are not so poor (7). p16 is tight-binding protein for CDK4 and negatively regulates the cell cycle through a specific inhibition of the cyclinCDK4 complex activity. In a condition of p16 gene deletion or its dysfunction, the gap phase (G) in the cell cycle may uncontrollably proceed to synthesis (S) and mitosis (M), and then the cell proliferation will be accelerated. Therefore, the majority of pancreas cancer cells might be in a state in which the cell cycle is unregulated because of deletions or mutations of the p16 gene. These data suggest that this impaired gene function might be a major factor of the uncontrolled cell proliferation and tumor malignancy of human pancreas cancer (8). In this study, we constructed the adenovirus p16 expression vector and then investigated whether it could control the proliferation of pancreas cancer MIAPaCa-2 cells in which the p16 gene was deleted and discussed its significance for the adjuvant therapeutic method in the surgical treatment of pancreas cancer. MATERIALS AND METHODS LacZ Expression. Prior to p16 expression vector experiment, we set up a LacZ expression experiment to investigate whether the adenovirus vector works well enough to express the gene in pancreas cancer MIAPaCa-2 cells. Adenovirus LacZ expression vector (Adex1CALacZ) was a generous gift from Dr. I. Saito (The Institute of Medical Science, The University of Received 6/21/99; revised 8/31/99; accepted 9/2/99. 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. 1 Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan. 2 To whom requests for reprints should be addressed, at Second Department of Surgery, Chiba University School of Medicine, 1-8-1 Inohana, Chuoh-ku, Chiba 260-8670, Japan. Phone: 81-43-226-2109; Fax: 8143-226-2113; E-mail: [email protected]. 3 The abbreviations used are: MTS1, multiple tumor suppressor 1; CDK, cyclin-dependent kinase 4; X-gal, 5-bromo-4-chloro-3-indolyl-b-Dgalactopyranoside; MOI, multiplicity of infection. 4182 Vol. 5, 4182–4185, December 1999 Clinical Cancer Research Research. on December 31, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Tokyo, Tokyo, Japan) (9). We introduced Adex1CALacZ into MIAPaCa-2 for 60 min and washed the cells twice with PBS, after which the cells were incubated. For cell staining, 3 days after infection, the cells were washed twice with PBS again, fixed with 0.25% glutaraldehyde, and stained with 0.1% X-gal (10). Construction of Adenovirus p16 Expression Vector. As the first step of construction of adenovirus p16 expression vector, we inserted p16 cDNA into the SwaI site of a cassette cosmid containing a nearly full-length adenovirus type 5 genome with E1 (nucleotides 454-3328) and E3 (nucleotides 28592–30470) deletions through an in vitro package (Stratagene). The inserted p16 sequence was cut out from p16 cDNA by a PCR procedure as the size of three bases upstream to the starting codon and 31 bases downstream from the stop codon, fitting Kozak’s rule (11), to improve gene expression. In the cassette cosmid into which p16 had been inserted, the p16 gene could be of right or leftward orientation. The efficiency of expression was known to be better in leftward orientation; therefore, we selected the leftward-directed cassette cosmid by restriction-enzyme digestion (9). To produce a recombinant adenovirus (AdexCACSp16), the expression cosmid and adenovirus DNA-terminal protein complex were cotransfected into 293 cells by calcium phosphate precipitation after the digestion of adenovirus DNA-terminal protein complex by EcoT22I (TaKaRa, Otsu, Japan). Recombinant adenovirus was isolated and expanded in 293 cells, and then the viral solutions were stored at 280°C. The virus titer was determined by plaque assay using 293 cells as described (12). Cell Culture and Infection of Adenovirus Vectors. The human pancreas cancer cell line MIAPaCa-2 was obtained from the Japanese Cancer Research Resources Bank. We also used human embryonic kidney cell line 293 cells, which were obtained from American Type Culture Collection, for the adenovirus recombination. These two cell lines were cultured in DMEM containing 10% fetal bovine serum. The pancreas cancer cell MIAPaCa-2 was seeded and cultured in 96-well plates at a density of 2 3 10 cells/1.0 ml of medium supplemented with 10% fetal bovine serum for 24 h. Immediately before infection, the culture medium was removed from the wells, and then the suspension of adenovirus vectors [AdexCACSp16, Adex1CALacZ, and Adex1w1 (control virus)], at MOI of 30, was plated onto the cells of the monolayer. The cell-vector contact as infection was kept for 1 h, and subsequently the cells were incubated by adding DMEM medium. Northern Blot Hybridization. We evaluated the activity of adenovirus p16 expression vector to express p16 gene mRNA in the pancreas cancer cell line MIAPaCa-2 by Northern blot hybridization. Total RNA was obtained from MIAPaCa-2 cells dishes at 24 h incubation after infection. The RNA extraction was carried out following the guanidinium thiocyanate/CsCl procedure involving high-speed centrifugation (13). The cells were mixed with guanidinium thiocyanate solution and homogenized at 4°C. Each 1-ml homogenate had 0.4 g of CsCl added to it and was then layered onto a 0.6-ml cushion of 5.7 M CsCl with 0.1 M EDTA. This solution were centrifuged at 45,000 rpm for 12 h at 20°C, and then RNA pellets were dissolved in a buffer of 10 mM Tris-HCl, 5 mM EDTA, and 1% SDS and then purified through the procedure using chloroform/n-butyl alcohol and ethanol. The Northern blots hybridization was prepared with 10 mg of total RNA obtained from MIAPaCa-2 cells. The electrophoresis of RNA was performed on 1.0% agarose gels prepared in MOPS buffer (0.02 M morpholinopropanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0) containing 2.2 M formaldehyde (14). After the identification of 28S and 18S RNA stained by ethidium bromide, the denatured RNA was transferred to the nylon membrane filters (Amersham International, Buckinghamshire, United Kingdom). We used p16 cDNA as the p16 gene probe (15). The filters were incubated with this Plabeled probe for 15 h in a solution containing 50% formamide, 53 saline and sodium citrate, 53 Denhardt’s solution, and 20 mg/ml denatured salmon sperm DNA after prehybridization. The filter was washed in 23 SSC containing 0.1% SDS at 60°C and then exposed to X-ray film. Effect of AdexCACSp16 on Cell Proliferation. The MIAPaCa-2 cells were seeded in 48-well plates and infected with adenovirus vectors (AdexCACSp16 and Adex1w1) at MOI of 30. The number of the living cells was counted before infection, on the first day, third day, fifth day, and seventh day after infection. Thereafter, we compared the number of cells between the groups of AdexCACSp16, Adex1w1, and no virus by statistical analyses, and then we assessed the effect of the p16 expression vector on MIAPaCa-2 cell proliferation. As a statistical analysis, we used ANOVA corrected for repeated measures to analyze the data for the difference between the three groups. Post hoc analysis was performed at individual time points using Tukey’s test with a computer statistical package. P , 0.05 was considered significant.