人体胰腺癌细胞株PANC-1中四环素可诱导的周期素D1表达抑制系统的建立
作者：王建承 张卓 林谋斌 梁鲁
【摘要】 ［目的］ 在人体胰腺癌细胞株PANC-1中建立一个四环素可调控周期素D1表达系统，研究抑制周期素D1对胰腺癌细胞的影响。［方法］ 反义周期素D1质粒通过两次稳定转染进入胰腺癌细胞株PANC-1细胞中，其表达调控系统采用Tet-Off系统（四环素调控系统）。通过抑制周期素D1表达对PANC-1细胞生长、集落形成能力以及周期素蛋白表达的影响，评价此系统的可调控性和有效性。［结果］ 通过第一次转染pTet-Off质粒，选择两个最佳表达克隆行第二次转染pTRE-反义周期素D1质粒，并通过免疫印记测定挑选出能在Tet-Off系统中最有效地表达反义周期素D1的克隆。通过Tet-Off系统对反义周期素D1的调控，发现周期素D1的表达抑制可明显地抑制胰腺癌细胞生长和集落能力，并可导致胰腺癌细胞形态学改变。其抑制作用与四环素调控浓度和时间有关。 ［结论］ 此研究在PANC-1胰腺癌细胞株中建立了一个高效、可诱导的反义周期素D1的表达系统。通过这个系统的建立，可进一步在体内和体外研究周期素D1的抑制对胰腺癌细胞的影响，并可结合其它手段如化疗来探讨联合治疗在临床的潜在应用价值。
【关键词】 周期素D1 反义Tet-Off系统 胰腺肿瘤 PANC-1
Pancreatic cancer is the fourth to fifth leading cause of cancer death in the world with an overall 5-year survival of only 0.4%[1]. Overexpression of growth factors in conjunction with deregulated cyclin
D1 expression has been proposed to contribute to the
loss of cell cycle control and to enhance tumorigenesis[2]. A study of 82 pancreatic cancers demonstrated overexpression of cyclin D1 protein in 65% of the tumors, and this was associated with shorter survival for these patients[3]. In another study patients with high levels of cyclin D1 mRNA had a median survival of 6.5 months, whereas patients with low levels had a median survival of 15.5 months[4].
The aim of this project was to establish human pancreatic cancer cells that harbor CD1 antisense cDNA in a tetracycline inducible vector system that can be turned on and off depending on the surrounding tetracycline concentration. And with these cells the effects of inhibition of expression of CD1 were also explored.
1 MATERIAL AND METHODS
1.1 Material
PANC-1 human pancreatic cancer cells from American Type Culture Collection; pTet-Off, pTRE-Luc and pTK-Hyg from Clontech Laboratories Inc U.S.; plasmid maxi kit and QIAprep@Spin mini prep Kit from Qiagen; lipofectamine from Gibco; luminometer and the dual-luciferase reporter assay system from Promega; Mouse nonoclonal anti-cyclin D1 antibody (DCS-6) from Neomarker Inc; anti-mouse IgG horseradish peroxidase linked whole antibody (from sheep), anti-β-actin antibody; electrochemiluminescence (ECL) solution and Hybond-P membranes from Amersham Biosciences Germany; DNA ladder (1 Kb plus), pH5αTM chemically competent cells from Invitrogen.
1.2 Methods
1.2.1 Cell Culture
PANC-1 cells were grown in DME supplemented with 10% fetal bovine serum, penicillin G (100 units/ml), and streptomycin (100 mg/ml) termed complete medium. Cells were maintained in monolayer culture at 37℃ in humidified air with 5% CO2. The medium for cell lines containig a neomycin resistance gene was supplemented with 1.2 mg/ml G418 and 0.2 mg/ml for containing a hygromycin resistance gene and termed selection medium. DME supplemented with 0.1% BSA, 5mg/L transferrin, 5mg/L selenium, penicillin G (100 units/ml), and streptomycin (100 mg/ml) termed serum free medium.
1.2.2 Transformation and Plasmid Preparation
The pTet-Off system consisted of the following plasmids: a regulator plasmid (pTet-Off), a luciferase response plasmid (pTRE-Luc), a hygromycin resistant plasmid (pTK-Hyg) and a response plasmid including full-length human cyclin D1 antisense cDNA (pTRE-CD1AS).
E. coli cells (DH5α) were used for transformation with plasmids. After colony formation mini preparations were performed first, followed by maxi preparations after certification of the authenticity of the plasmid with restriction endonuclease digests as shown for pTet-Off, pTRE-Luc and pTK-Hyg, respectively.
To prepared pTRE-CD1AS the 1.1-kb EcoRI/Bam HI full-length human cyclin D1 cDNA was subcloned in its antisense orientation into pTRE vector.
1.2.3 Transfection
1.2.3.1 First Stable Transfection with pTet-Off Vector
Cells were cultured in 10cm dishes to a density of 70%~80%. Then 40ml lipofectamine and 40mg pTet-Off plasmid DNA was added to two tubes with 3ml serum free medium separately. The two solutions were homogenized before they were then mixed together. The mixture was then incubated for 30 minutes at room temperature. Cells were washed in the meantime two times with 1×PBS and one time with serum free medium. Then, the homogenized mixture of lipofectamine and the pTet-Off plasmid DNA were added to the cells which were incubated for 5 hours at 37℃. After that an equal volume of complete medium containing 20% FBS was added for an additional 20 hours. Next, cells were washed two times with 1×PBS and two times with complete medium. After incubating for another 24 hours, cells were split into eight 10cm dishes and incubated in selection medium (1.2mg/ml G418). Selection medium was changed weekly. After 2 weeks colonies formed. The visible colonies were picked individually with sterile cotton tips wetted with trypsin. The picked colonies were transferred into 24-well plates including 1ml selection medium per well.
1.2.3.2 Transient Transfection with pTRE-Luc
Cells were cultured in 10cm dishes to a density of 70%~80%. On the following day, 40ml lipofectamine and 40mg pTRE-Luc plasmid DNA was added to separate tubes with 3ml serum free medium. The two solutions were homogenized before they were mixed together to make the transfection solution. The mixture was then again incubated for 30 minutes at room temperature. Cells were washed two times with 1×PBS and one time with serum free medium. The mixture was homogenized again and added to the cells. Cells were then incubated overnight at 37℃. On the following day, cells were washed one time with 1×PBS and transferred into 24-well plates with complete medium (every clone into 4 wells). Tetracycline (2mg/ml) was added to 2 of the 4 wells of each clone. Cells were then incubated 24 hours at 37℃ and collected for luciferase activity assay.
1.2.3.3 Second Stable Co-transfection with pTRE-cyclin D1 Antisense and pTRE-Hyg
Cells were cultured in 10cm dishes to a density of 70%~80%. After that 40ml lipofectamine, 40mg pTRE-CD1AS and 2mg pTRE-Hyg plasmid DNA were mixed and incubated in 6ml serum free medium for 30 minutes at room temperature. Cells were then washed two times with 1×PBS, one time with serum free medium and incubated with the transfection solution overnight in the presence of tetracycline (2μg/ml). On the next day, cells were washed two times with 1×PBS and two times with complete medium and then incubated with complete medium in the presence of tetracycline (2μg/ml).
Cells were allowed to grow to about 80% confluence and then split into eight 10cm dishes followed by incubation with complete medium including hygromycin (0.2mg/ml) and tetracycline (2μg/ml). Medium was changed once a week including the respective addition. After 4 weeks colonies had formed and were picked individually as described above. Colonies were transferred into 24-well plates in 1ml selection medium including tetracycline (2μg/ml). When cells reached confluence they were transferred into flasks in selection medium including G418(1.2mg/ml), hygromycin (0.2mg/ml) and tetracycline (2μg/ml).
1.2.3.4 Dual-luciferase Reporter Assay
Cells were collected and processed according the protocol described with Dual-Luciferase Reporter Assay System kit. A luminometer was used to measure the activity.
1.2.3.5 MTT-Assay
75,000 cells/ml were seeded in 200ml complete medium in 96-well plates. On the next day, medium was removed without removing cells. Cells were washed with 1×PBS (200ml) and then 200ml medium in absence or presence of desired additives was added to each well. Every 24 hours medium was changed. To initiate the assay, 25ml MTT stock-solution (5%) was added in each well. Cells were incubated for additional 4 hours prior to the removal of the medium. The developed dye crystals were dissolved in 100ml acidic isopropanol (isopropanol with 0.04 N hydrogen chloride) for 15 minutes. After that, the optical density values were measured using a microplate reader at 570nm. The value at 650nm was subtracted as background.
1.2.3.6 Soft Agar-assay
Medium A (15% fetal bovine serum, 1mM sodium-pyruvate, 8μg/ml L-serine, 100units/ml penicillin, 100μg/ml streptomycin, 11% tryptic soy broth, 16 μg/ml L-asparagine with Mc Coys′5 A,) and medium B (15% fetal bovine serum, 2 μg/ml insulin, 1% vitamine C, 100units/ml penicillin, 100μg/ml streptomycin, 2mM L-glutamine, 1%L-asparagine with RPMI medium) were prepared first. 1% agar solution was melted by microwaving and added to medium A (1∶5 v/v) to make base agar. Then, 1ml was poured into per well of 3.5cm dishes and kept at room temperature until it became solid.
In the same time, cells were trypsinized and diluted to 3.33×105 cells/ml in medium B. Cells were then incubated in a water bath at 37°C and 1% agar solution (50°C) was added to the cell suspension (1∶2.3 v/v) including the desired treatment agents, mixed and poured (1ml per well) as upper agar. Later the agar plates were incubated at 37℃ in humidified air with 5% CO2. After 5 days, cells were fed with 0.5ml feeding solution (1% agar: medium B 1∶2(v/v)) per well. After 10~14 days 50μl 5%MTT solution was added to each plate. The next day stained colonies were counted under a microscope and photographed.
1.2.3.7 Immunoblotting
Exponentially growing cells (40%~50% confluent) were lysed and protein concentration of each cell lysate was pdetermined by BCA Protein Assay Reagent kit. Cell lysates were then subjected to 12% SDS-PAGE, run with 70V for 30 minutes and then with 160V for 90 minutes. Electrophoresed proteins were electrotransferred from the gel to Hybond-P membranes using 110V for 60 minutes. After blocking in 5% milk in 1×TTBS, the membranes were blotted with a highly specific mouse monoclonal anti-cyclin D1 antibody (DCS-6, 1∶500) and with a secondary anti-mouse horseradish-conjugated antibody (1∶1000). Bound antibodies were visualized using enhanced chemiluminescence.
To confirm equal loading, membranes were stripped for 30 minutes at 50℃ in buffer and re-probed with an anti-β-actin antibody.
1.2.3.8 Flow Cytometry
Cells were harvested by scraping and washed three times with buffer solution (cycle testTM plus DNA reagent kit). Cells were then lysed and stained with propidium iodide staining solution according to instructions of the kit. Samples were run on the flow cytometer (FACSScan, Becton Dickinson) and data were collected and analyzed by CELLQuest software (Becton Dickinson).
1.3 Statistical Analysis
Statistical analysis was performed with SPSS software. Results are expressed as Mean ±SD or as Mean±SEM. When indicated, Student’s t test was used for statistical analysis with P<0.05 as the level of significance.
2 RESULTS
2.1 Establishment of the pTRE-CD1AS-Tet-Off System in PANC-1 Cells
Primary transfection with pTet-off
Overall, 24 clones were randomly picked and propagated as individual clones. After further expansion of the clones, inducibility of the pTet-Off regulator plasmid was tested using a transient transfection with the pTRE-Luc response plasmid. The best clones regulated by tetracycline were those having the highest increase in fold of luciferase activity when tetracycline was removed compared to those with tetracycline. All 24 clones were screened by luciferase readout. Among them clones p19 and p10 showed the highest inducibility after removal of tetracycline (Figure 1).
Figure 1. Results of Luciferase readout: PANC-1 cells were transiently transfected with pTRE-Luc. 24 hours after transfection, in half of the wells tetracycline (2μg/ml) was added. Cells were collected 24 hours later and tested by luciferase readout (n=2). Clones were named p1 to p24. Data was expressed as increased fold of different clones. The remaining clones with low or no increase in luciferase readout were only tested once (n=2).
Secondary co-transfection with pTRE-CD1AS and pTK-Hyg
Clone p19 was used for the secondary transfection as they showed the highest inducibility of luciferase activity by 24 folds. After about 3~4 weeks when visible clones formed, they were picked individually and from then on cultured as individual cell clones in selection medium containing tetracycline.
2.2 Characterization of Cyclin D1 Antisense in Expression in PANC-1 Cells
To investigate the function of the CD1AS system, clones were subjected to immnunoblotting to determine the cyclin D1 protein level. The results revealed that cyclin D1 protein expression decreased when cells were treated without tetracycline for 3 days (Figure 2A). On day 1 and day 2, there was no obvious inhibition of expression of cyclin D1 protein and on day 3, its expression was decreased 50.9% as determined by densitometric analysis (Figure 2B). One of 27 clones named p19-T from parental clone p19 was selected because of its good inducibility of the system.
2.3 Anchorage-dependent Growth Assay
Selected clone with good function of the CD1AS-TET system was investigated on cell growth by the MTT growth assay. The inhibition was dose-dependent and 0μg/ml of tetracycline resulted more inhibition of cell growth compared to 0.01μg/ml (P<0.05). Inhibition of growth was also time-dependent. On day 1 and day 2 after removal of tetracycline there were no marked effects, while on day 3 and day 4 cell growth was markedly inhibited (Figure 3,P<0.01). Cell growth was inhibited by 14.2% and 34.6% on day 3 and day 4, respectively, in the presence of 0.01μg/ml tetracycline and by 35.4% and 54.5% on day 3 and day 4, respectively, in the absence of tetracycline in p19-T cells.
The results of the MTT growth assay were confirmed by cell counting. For clone p19-T the cell number decreased from day 1 to day 4 in the absence of tetracycline. The cell number compared to control was reduced by 27.2%, 38.0%, 54.9% and 65.1% on day 1, 2, 3 and 4, respectively (Figure 4).
2.4 Morphological Characters
The results revealed that cells remained their growth ability and morphology after 3 days treated with complete medium in the presence of tetracycline. In contrast, when cells were cultured in the absence of tetracycline for 3 days, they lost their normal shape and shrank with decrease of density.
2.5 Anchorage-independent Growth Assay
The colony-forming ability for clone p19-T was inhibited when cultured in the absence of tetracycline compared to cells cultured in the presence of tetracycline. The number of formed colonies in both clones treated without tetracycline was markedly diminished compared to clones treated with tetracycline.
2.6 Effects of Cyclin D1 Antisense on Cell Cycle
The effect of CD1AS on cell cycle distribution next was investigated. Cell cycle analysis was performed for cells cultured in the presence and absence of tetracycline (Table 1). The results revealed that on day 1 after removal of tetracycline, there was no change in distribution of the cell cycle compare to the controls. In contrast, on day 2 and day 3, an increase of the fraction of cells in G0~1 and a decrease of the fraction of cells in S phase were seen. The percentage of cells in G0~1 compared to control on day 2 was 60% versus 54% while on day 3 was 56% versus 48%. FACS analysis also revealed that the percentage of cells in G2~M on day 2 and day 3 were decreased. The percentage of cells in G2~M compared to control on day 2 was 21% versus 29% while on day 3 was 21% versus 31%. Furthermore, the percentage of cells in pre-G0~1 on day 3 was increased compared to control, with 7% versus 3%.
3 DISCUSSION
Pancreatic cancer is an aggrsssive cancer that usually present at a late stage with poor prognosis. It seems to be important to aim at developing new strategies to inhibit pancreatic cancer cell growth while enhancing its sensitivity to multimodal therapy including chemotherapy and/or radiation.
Cyclin D1 with its catalytic partners CDK4 and CDK6 is a critical modulator of cell cycle progression through G1 and appears to have an important role in neoplastic transformation. Elevated cyclin D1 mRNA levels have been reported in several of these malignancies[3] in association with shortened G1 phase, decreased cell size, and reduced dependency on mitogens.
Clinically, increased cyclin D1 levels have been correlated with decreased survival of patients with cancers of the pancreas[4,5]. Inhibition of cyclin D1 by antibody microinjection or by CD1AS has shown to prevent normal fibroblasts from entering the S phase of the cell cycle and markedly inhibiting the proliferation of human colon[6] and pancreatic cancer cells[7]. Furthermore, transduction of the CD1AS into preformed subcutaneous tumors in athymic mice resulted in inhibited tumor growth and prolonged animal survival[8].
The Tet-Off gene expression system was first described by Gossen & Bujard. In the Tet-Off system, gene expression is turned on when tetracycline is removed from the culture medium. The system permits gene expression to be tightly regulated in response to varying concentrations of tetracycline. Through double stable transfection of the regulator plasmid pTet-Off and the response plasmid pTRE-CD1AS into PANC-1 cells, we established an inducible inhibition of the cyclin D1 system by tetracycline that was proven to be efficient of inhibiting cyclin D1 protein expression when tetracycline was removed from the medium.
Our study revealed that gene expression was restrained even at very low concentration of tetracycline (0.01 μg/ml). The repression concentration of tetracycline was lower than previously reported[9]. The results also revealed that gene expression was dose dependent on tetracycline, and that this system can also be applied for in vivo studies. The present study revealed that after removal of tetracycline, the corresponding time of gene repression began at about 48 hours assessed by immunoblot analysis, cell growth and cell cycle assays. This result was consistent with other studies showing that the usual time to get maximal expression after removal tetracycline in the Tet-off system was 48 hours[9].
Thus, the PANC-1 cells constructed with the pTet-Off system and pTRE-CD1AS is a well-established cell line, and can be regulated by tetracycline with high sensitivity, inducibility and fast response for CD1AS expression. To our knowledge, this is the first established cell line constructed with Tet-Off and CD1AS.
Inhibition of cyclin D1 by expression of CD1AS cDNA induced by the Tet-Off system could suppress pancreatic cancer cell growth both in vitro and in vivo. It was reported that reduction in cyclin D1 mRNA occurred by 24 hours and in protein by 48 hours after treatment of ovarian cancer cells with AS Oligonucleotides in vitro[10]. Our results suggested that CD1AS effects started after 24 hours and demonstrated marked cell growth inhibition from 48 hours on after tetracycline was removed in vitro, while protein expression of cyclin D1 was markedly decreased after 72 hours. This could be explained by the time-lapse of the Tet-Off system after removal of tetracycline[11]. By anchorage-independent growth assays, it could be demonstrated that inhibition of cyclin D1 significantly suppressed the ability of colony formation of pancreatic cancer cells. All these results support the fact that cyclin D1 is an important factor for cell proliferation and tumorigenicity[8].
Our results revealed that CD1AS could retard cell growth in phase G0~1 of cell cycle. There was also a tendency of increasing percent of pre-G0~1 phase, when expression of CD1AS lasted for 3 days. Cells losing their normal shape, shrinking and decrease of cell density were observed in our study by microscopy when tetracycline was removed for 3 days. Whether CD1AS only results in preventing cell progression from G0~1 to S phase or also leads to apoptosis is under investigation. These studies suggested that inhibition of cell growth in vivo and in vitro might occur due to cell cycle inhibition as well as apoptosis.
The approach to suppress cyclin D1 by CD1AS expression and thereby inhibit cancer cell growth is promising. With the establishment of an inducible inhibition of cyclin D1 by the tetracycline system in PANC-1 cells which was established in this study, it is now possible to investigate the effects of combining of cyclin D1 suppression and the effects of chemotherapeutic agents both in vitro and in vivo.
【】
[1] Bramhall SR, Allum WH, Jones AG, et al. Treatment and survival in 13,560 patients with pancreatic cancer in the West Midlands, and incidence of the disease, an epidemiological study[J]. Br J Surg, 1995, 82(1): 111-115.
[2] Motokura T,Arnold A.Cyclins and oncogenesis[J]. Biochim Biophys Acta, 1993, 1155(1): 63-78.
[3] Hall M,Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer[J]. Adv Cancer Res, 1996, 68: 67-108.
[4] Kornmann M, Ishiwata T, Itakura J et al. Increased cyclin D1 in human pancreatic cancer is associated with decreased postoperative survival[J]. Oncology, 1998, 55(4): 363-369.
[5] Gansauge F, Gansauge S, Schmidt E, et al. Prognostic significance of molecular alterations in human pancreatic carcinoma—an immunohistological study[J]. Langenbecks Arch Surg, 1998, 383(2):152-155.
[6] Arber N, Doki Y, Han EK, et al. Antisense to cyclin D1 inhibits the growth and tumorigencity of human colon cancer cells[J]. Cancer Res, 1997, 57(8): 1569-1574.
[7] Kornmann M, Danenberg KD, Arber N, et al. Inhibition of cyclin D1 expression in human pancreatic cancer cells is associated with increased chemosensitivity and decreased expression of multiple chemoresistance genes[J]. Cancer Res, 1999, 59(14): 3505-3511.
[8] Uto H, Ido A, Moriuchi A, et al. Transduction of antisense cyclin D1 using two-step gene transfer inhibits the growth of rat hepatoma cells[J]. Cancer Res, 2001,61(12):4779-4783.
[9] A-Mohammadi S,Alvarez-Vallina L,Ashworth LJ, et al. Delay in resumption of the activity of tetracycline-regulatable promoter following removal of tetracycline analogues[J]. Gene Ther, 1997, 4(9): 993-997.
[10] Cagnoli M, Barbieri F, Bruzzo C, et al. Control of cyclin D1 expression by antisense oligonucleotides in three ovarian cancer cell lines[J].Gynecol Oncol,1998,70(3):372-377.
[11] Rennel E, Gerwins P. How to make tetracycline-regulated transgene expression go on and off[J]. Anal Biochem, 2002, 309(1): 79-84.
