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Expression of Fas-estrogen receptor fusion protein induces cell death in pancreatic cancer cell lines
Cancer Letters 116 (1997) 53–59
Expression of Fas-estrogen receptor fusion protein induces cell death in pancreatic cancer cell lines Yoshiya Kawaguchi a,*, Hirohide Takebayashi b, Akira Kakizuka b, Shigeki Arii a, Masayuki Kato a, Masayuki Imamura a a
Department of Surgery and Surgical Basic Science, Kyoto University, 54 Shogoin-Kawaracho, Sakyoku, Kyoto 606, Japan Department of Pharmacology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyoku, Kyoto 606, Japan
b
Received 28 November 1996; revision received 10 February 1997; accepted 21 February 1997
Abstract Recently, a novel system to induce apoptosis was reported. Fusion protein between Fas and the ligand-binding domain of the estrogen receptor (MfasER) could cause apoptotic cell death in an estrogen-dependent manner on murine fibrosarcoma L929 cells and human cervical carcinoma HeLa cells [1]. To investigate the application of this system to the gene therapy of pancreatic cancer, we introduced MfasER cDNA to six cell lines. Transiently expressed MfasER could cause cell death in all the cell lines tested. Furthermore, stably MfasER-expressing cells showed DNA fragmentation as early as 2 h and completely died in 48 h in the presence of estrogen. Combined with effective methods to introduce genes to pancreatic cancer selectively, MfasER would be a good tool for the gene therapy of pancreatic cancer in the future. 1997 Elsevier Science Ireland Ltd. Keywords: Fas; Estrogen receptor; Fusion protein; Gene therapy; Pancreatic cancer
1. Introduction Pancreatic cancer is one of the most difficult diseases to be cured. The majority of this disease is invasive ductal adenocarcinomas that expand very rapidly and form metastatic lesions. Despite the intensive therapies including surgical operation, chemotherapy or radiation therapy, clinicians are faced with disappointing outcomes in most cases [2–5]. The breakthrough to this difficult disease has long been waited for but not seen yet. One possible approach is gene therapy, introducing * Corresponding author.
exogenous genes involved in cell death machinery. Among them, Fas is a type I membrane protein which mediates apoptotic death signal [6]. Recently, a new method to induce apoptosis was reported. Fas fusion protein with the ligand binding domain of the estrogen receptor (MfasER) stably expressed in murine fibrosarcoma L929 cells or human cervical carcinoma HeLa cells could cause apoptotic cell death in an estrogen-dependent manner [1]. In this report, to investigate the application to the gene therapy of pancreatic cancer, we examined the effect of MfasER on six cell lines from human pancreatic cancers and demonstrate that MfasER can induce cell death in all the cell lines tested.
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved P II S0304- 3835 (97 )0 4751- 4
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Y. Kawaguchi et al. / Cancer Letters 116 (1997) 53–59
2. Materials and methods 2.1. Plasmids Construction of MfasER cDNA is almost the same as previously reported [1]. Briefly, cDNA for the transmembrane and intracellular domain of mouse Fas (amino acids 135–305), and cDNA for the ligand binding domain of rat estrogen receptor (amino acids 286–600) are ligated into the expression vector pEF-BOS-bsr with translation initiation site followed by the influenza virus hemagglutinin (HA) epitope, which is recognized by a monoclonal antibody, 12CA5 (Boehringer Mannheim). HA epitope is added at the C-terminal of the fusion protein. Resulting plasmid was named pEF-BOS-bsrMfasER. 2.2. Cell culture Human pancreatic adenocarcinoma cell lines KMP2, 3, 4 and 5 were independently established from resected specimens operated in Kyoto University Hospital (manuscript in preparation; established by Drs. M. K. and Y. Shimada, Kyoto University). MIAPaCa2 and Panc1 were supplied by A.T.C.C. All cell lines were maintained in DMEM supplemented with 10% bovine calf serum (HyClone). 2.3. Transient expression of MfasER and luciferase assay All transfections were carried out with six samples and at least three independent experiments were performed to confirm the reproducibility. Transfections were performed with LipofectAMINE (Gibco BRL) [7]. Cells (2 × 105) were plated on six-well plates and cultured until 50% confluent in DMEM supplemented with 10% serum. Culture medium was removed and cells were washed twice with optiMEM (Gibco BRL). Cells were cotransfected with 1 mg of pEF-BOS-bsr-MfasER and 0.2 mg of pCMXLUC, or 1 mg of pEF-BOS-b-gal and 0.2 mg of pCMX-LUC (as control). After 4 h, transfection medium was replaced with DMEM supplemented with 10% serum, and 12 h later, estradiol (E2) was added at the concentration of 10−9 M. After 33 h of E2 treatment, cells were washed with PBS and har-
vested with cell scraper in 1 ml of PBS. Cells were collected by the centrifugation (15K, 15 s), and resuspended in 100 ml of 0.25 M Tris–HCl (pH 7.5) with Vortex mixer for 10 min. Proteins were eluted by three cycles of freeze-thaw method; each cycle consisted of 2 min in dry ice-cold ethanol and 2 min in water bath at 37°C. After the centrifugation (15K, 10 min), supernatants were collected. Luciferase activities were measured for 30 s with Lumat LB 9501 (Berthold, Germany) as described [7,8]. 2.4. Establishment of stably MfasER-expressing clones The stably MfasER-expressing cells were established as follows: KMP4 and MIAPaCa2 cells were plated on 9 cm dishes, cultured until 50% confluent and transfected with the linearized MfasER expression plasmid by lipofection method. Cells were detached from plates with trypsin and divided into three 9 cm dishes. After selection with blasticidin S (Funakoshi, Japan) at the concentration of 10 mg/ml for 3 weeks, the cells were cloned. 2.5. Assay for cell death Cells were photographed through a Nikon microscope at × 100, 24 h after E2 addition at 10−8 M. For quantification, cells cultured in 96-well plates were stained with crystal violet before and 6, 18, 24, 48 h after the addition of 10−8 M E2. The percentage of viable cells was calculated as described [6,9]. Low molecular weight DNA was purified as follows. Each cell line, plated on 15 cm dish plate, was cultured with or without E2 at 10−8 M for 2 h. Cells were harvested with cell scraper and centrifuged at 1K for 5 min. Collected cell pellets were suspended in 1 ml of PBS and transferred to 1.5 ml tube. After the centrifugation (2K, 5 min), cells were resuspended in 500 ml of 10 mM Tris–HCl (pH 7.5), 10 mM EDTA and 0.2% Triton X-100. Centrifuged at 9K for 10 min at 4°C, the upper aqueous phase was collected, underwent phenol extraction once and phenol-chloroform extraction once, and precipitated with ethanol. DNA pellets were washed with 70% ethanol and suspended in TE buffer. DNA samples were electrophoresed in 2% agarose gels, visualized by ethidium bromide staining and photographed.
Y. Kawaguchi et al. / Cancer Letters 116 (1997) 53–59
Fig. 1. Schematic structure of the Fas, estrogen receptor (ER), and Fas-estrogen receptor fusion protein (MfasER) [1]. The amino acid numbers of the original Fas and ER are shown above and below the schematics, respectively. S, signal peptide; TM, transmembrane domain; DD, death domain; HA, hemagglutinin epitope; LBD, ligand (estradiol) binding domain; DNA, DNA-binding domain.
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Vortex mixer for 15 min and boiled for 5 min. Cell lysates from 5 × 105 cells of parental KMP4 and transiently transfected samples, and 1.7 × 105, 1.7 × 104, 5.7 × 103, 1.9 × 103, 6.3 × 102 of KMP4-34 cells were separated on 10% SDS-polyacrylamide gel and transferred to PVDF membrane. The blot was reacted with the anti-HA monoclonal antibody, 12CA5 (Boehringer Mannheim) [1,7], and the expressed MfasER protein was detected by the alkaline phosphatase color detection system (Promega) for 30 min.
3. Results
2.6. Western blotting Parental KMP4 cells, stably MfasER-expressing KMP4-34 cells, and transiently MfasER-transfected KMP4 cells were harvested and the cell number counted. In transient transfection, samples were obtained 16, 22, and 49 h after transfection. Transfection procedure was the same as above (Section 2.3). At the same time, transfection efficiency was evaluated by X-gal staining of the control transfection. Cells were lysed in 2 × SDS gel-loading buffer with
Schematic structure of MfasER is shown in Fig. 1. The carboxyl-terminal portion of Fas is fused with the ligand binding domain of the estrogen receptor. The transmembrane and death domains of Fas are reported to be essential for mediating apoptotic signal of MfasER [1]. To investigate the cell death effect of MfasER quantitatively, we underwent cotransfection of MfasER expression vector and luciferase expression vector (MfasER+ Luc) to six pancreatic cancer cell lines;
Fig. 2. Relative luciferase activity on transient coexpression of MfasER and Luc (lanes 1 and 2) or b-gal and Luc (lanes 3 and 4 as controls) in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of E2. Relative luciferase activity represents the relative viability of transfected cells (details in the text). From the representative experiment, mean values and standard deviations are shown (n = 6 in each transfection).
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Fig. 3. Estrogen-induced cell death in stably MfasER-expressing cells ( × 73). Cells are healthy in normal medium (B, KMP4-34; D, MIA351), but become round-shaped and detaching from the dishes 24 h after E2 addition (A, KMP4-34; C, MIA35-1).
it was known that the luciferase activities parallel the number of live cells expressing the transfected cDNAs [7]. Luciferase activities of MfasER+ Luc cotransfectant were compared to that of b-gal+ Luc cotransfectant, since b-galactosidase is widely used and believed to show no toxicity. As shown in Fig. 2, transient expression of MfasER induced significant decrease in luciferase activity in the presence of 10−9 M E2 (lane 1), demonstrating that transiently expressed MfasER could cause cell death in all the cell lines tested in the presence of estradiol. This effect was not due to the cell toxicity of estradiol, since the luciferase activities were not influenced by the addition of E2 in control b-gal transfections (lanes 3 and 4). Furthermore, in all the cell lines except MIAPaCa2, decreased luciferase activity was observed even in the absence of E2 (lane 2). This suggests that expressed MfasER has its own constitutive activity (see Section 4). For further investigation, we obtained cell clones that stably express MfasER. Among them, KMP4-34 established from KMP4 and MIA35-1 from MIAPaCa2 were selected. Parental KMP4 and MIAPaCa2 cells showed no morphological changes by the addi-
tion of E2 (data not shown). As shown in Fig. 3, stably MfasER-expressing clones KMP4-34 and MIA35-1 demonstrated clear phenotypic changes in the pre-
Fig. 4. Quantitative analysis of cell viability, evaluated by the crystal violet assay. In the presence of E2, stably MfasER-expressing cells die completely in 48 h.
Y. Kawaguchi et al. / Cancer Letters 116 (1997) 53–59
Fig. 5. Agarose gel electrophoresis of low molecular weight DNA. Stably MfasER-expressing KMP4-34 and MIA35-1 cells show clear DNA fragmentation 2 h after E2 addition (lanes 1 and 2, respectively), but not in the absence of E2 (lanes 3 and 4, respectively). On the other hand, parental KMP4 and MIAPaCa2 cells do not show DNA fragmentation in the presence (lanes 5 and 6, respectively) nor in the absence (lanes 7 and 8, respectively) of E2.
sence of E2 (Fig. 3A,C, respectively), whereas each clone was healthy in the absence of E2 (Fig. 3B,D, respectively). The cells became round-shaped and shrunken, detaching from the culture plates. These morphological changes became apparent around 18– 24 h treatment of E2, and almost all cells were dead after 48 h. The cell viabilities were quantified by crystal violet assay and are shown in Fig. 4. In the absence of E2, there was no apparent difference in growth rates between parental cells and stable clones (data not shown), but in the presence of E2, stably MfasERexpressing cells died completely in 48 h. Furthermore, DNA fragmentation, a well known marker for apoptosis, was observed as early as 2 h after the addition of E2 in KMP4-34 and MIA35-1 cells (Fig. 5, lanes 1 and 2, respectively), but not apparent in the absence of E2 (Fig. 5, lanes 3 and 4, respectively). On the other hand, parental KMP-4 and MIAPaCa2 cells did not show clear DNA fragmentation regardless of E2 addition (Fig. 5, lanes 5, 6, 7 and 8).
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To evaluate the expression level of MfasER in transiently transfected cells and that in stably expressing cells, we undertook Western blotting using anti-HA monoclonal antibody, 12CA5 [1,7]. As shown in Fig. 6, expressed MfasER protein was detected in the cell lysates from 5 × 105 of transiently transfected cells harvested 16 and 22 h after transfection (lanes 7 and 8, respectively),with the stronger signal than that from 1.7 × 104 of stably-expressing KMP4-34 cells (lane 3). Considering that the transfection efficiency was 0.03%, evaluated by the X-gal-positive cells, 1.5 × 102 cells were expected to express MfasER in 5 × 105 of transiently transfected cells. Thus, compared to the stable clone, more than 100 times the amount of MfasER protein per cell is estimated to be expressed in transient experiment. Forty-nine hours after transfection, the amount of MfasER was decreased in the absence of estradiol (lane 9), indicating the cell death of the MfasER-overexpressing cells. In the presence of estradiol, MfasER became undetectable as early as 6 h after E2 addition (22 h after transfection), or 49 h after transfection (lanes 10 and 11, respectively). It means that estradiol promotes the cell death effect of over-expressed MfasER.
Fig. 6. Detection of expressed MfasER by Western blotting. 5 × 105 of parental KMP4 cells (lane 1), 1.7 × 105, 1.7 × 104, 5.7 × 103, 1.9 × 103 and 6.3 × 102 of stably MfasER-expressing KMP4-34 cells (lanes 2, 3, 4, 5 and 6, respectively), and 5 × 105 of transiently MfasER-transfected KMP4 cells (lanes 7, 8, 9, 10 and 11). In transiently transfected cells, cells were harvested 16, 22 and 49 h after transfection in the absence of estradiol (lanes 7, 8 and 9, respectively), or estradiol was added at 16 h after transfection then harvested 22 and 49 h after transfection (lanes 10 and 11, respectively). Expressed MfasER protein is indicated by the arrow. Note that MfasER is detected from as little as 6.3 × 102 of stably MfasER-expressing KMP4-34 cells (lane 6). Since the transfection efficiency was 0.03%, as counted by the X-gal-positive cells, 1.5 × 102 cells were expected to express MfasER in 5 × 105 of transiently transfected cells.
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4. Discussion In this report, we demonstrated that MfasER could cause cell death in all the pancreatic carcinoma cell lines tested. While there is little information about the expression of Fas in pancreatic cancers, anti-Fas stimulatory antibody CH11 [10] causes cell death in KMP5 and MIAPaCa2 but has little influence on KMP2, 3, 4, or Panc1 (our preliminary observations), suggesting that there is a heterogeneity in Fas-ligandmediated apoptosis among pancreatic cancer cell lines. Nonetheless, as demonstrated here, it should be noted that exogenously introduced MfasER could induce cell death even in the anti-Fas refractory cell lines, indicating that Fas-mediated intracellular signalling pathway is conserved in pancreatic cancer cell lines. Transiently expressed MfasER showed its constitutive activity in all the cell lines except MIAPaCa2, and cell death was promoted in the presence of estradiol (Fig. 2). It has been shown that ligand-dependent formation of oligomeric structure is important in the signal transduction of Fas and TNFR p55 [11,12]. Considering that death domains can multimerize by themselves [13], over-expressed MfasER might form oligomeric structure and transduce the death signal to its target molecules. Furthermore, there is a possibility that the addition of estradiol might facilitate the multimerization, since ER has been shown to form homodimers on its response elements [14]. Of course, the expression level of negative regulators such as bcl2 might possibly differ from cell lines to cell lines. It may account for why the constitutive activities of MfasER were different among cell lines and not apparent in MIAPaCa2. We also demonstrated that stably MfasER-expressing cell lines, KMP4-34 and MIA35-1, showed DNA fragmentation, a well known marker for apoptosis, as early as 2 h after E2 addition. It was far earlier than their phenotypic change. This result was consistent with the previous report [1]. The apoptotic features of estrogen-induced cell death were fully demonstrated in cloned MfasER-expressing L929 cells [1]: DNA fragmentation as early as 1 h after the addition of E2, apparent condensed nuclei by DAPI staining, and typical electron micrograph findings. The IC50 of estradiol was 3 × 10−10 M [1], capable of clinical use without severe side effects. Though we did not per-
form further confirmation in this report, it is easily imagined that MfasER-induced cell death in pancreatic cancer cell lines is also apoptosis. As demonstrated in the Western blotting, more than 100 times the amount of MfasER protein per cell is estimated to be expressed in the transient experiment than that in stable clone and causes constitutive activity in KMP4 cells. For the application to cancer gene therapy, if the MfasER protein is expressed in a cancer-specific manner, this constitutive activity is not a disadvantage. In other words, even the low expression of MfasER, that is, at least 1:100 amount of conventional transfection is capable of causing cell death in the presence of estradiol. It also supports the potential usefulness of MfasER in the gene therapy of pancreatic cancer. In conclusion, as we demonstrated here, MfasER could cause cell death in all the pancreatic carcinoma cell lines tested and its effect was strengthened by the addition of estrogen. For the practical application to the cancer gene therapy, however, many big problems remain to be solved: specific promoter must be cloned, effective methods to deliver exogenous genes must be developed and so on. Unfortunately, there is no effective method to selectively introduce genes to pancreatic cancer to date. Overcoming these obstacles, MfasER would be a good tool for the gene therapy of pancreatic cancer in the future.
References [1] Takebayashi, H., Oida, H., Fujisawa, K., Yamaguchi, M., Hikida, T., Fukumoto, M., Narumiya, S. and Kakizuka, A. (1996) Hormone-induced apoptosis by Fas-nuclear receptor fusion proteins: novel biological tools for controlling apoptosis in vivo, Cancer Res., 56, 4164–4170. [2] Conlon, K.C., Klimstra, D.S. and Brennan, M. (1996) Longterm survival after curative resection for pancreatic ductal adenocarcinoma, Ann. Surg., 223, 273–279. [3] Jessup, J.M., Steele, G. Jr., Mayer, R.J., Posner, M., Busse, P., Cady, B., Stone, M., Jenkins, R. and Osteen, R. (1993) Neoadjuvant therapy for unresectable pancreatic adenocarcinoma, Arch. Surg., 128, 559–564. [4] Shibamoto, Y., Manabe, T., Baba, N., Sasai, K., Takahashi, M., Tobe, T. and Abe, M. (1990) High dose, external beam and intraoperative radiotherapy in the treatment of resectable and unresectable pancreatic cancer, Int. J. Radiat. Oncol. Biol. Phys., 19, 605–611. [5] Bramhall, S.R., Allum, W.H., Jones, A.G., Allwood, A., Cummins, C. and Neoptolemos, J.P. (1995) Treatment and
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survival in 13560 patients with pancreatic cancer, and incidence of the disease, in the West Midlands: an epidemiological study, Br. J. Surg., 82, 111–115. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S.-I., Sameshima, M., Hase, A., Seto, Y. and Nagata, S. (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis, Cell, 66, 233–243. Ikeda, H., Yamaguchi, M., Sugai, S., Aze, Y., Narumiya, S. and Kakizuka, A. (1996) Expanded polyglutamine in the Machado-Joseph disease protein induces cell death in vitro and in vivo, Nature Genet., 13, 196–202. Kakizuka, A., Miller, W.H. Jr., Umesono, K., Warrell, R.P. Jr., Frankel, S.R., Murty, V.V.V.S., Dmitrovsky, E. and Evans, R.M. (1991) Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RARa with a novel putative transcription factor, PML. Cell, 66, 663–674. Itoh, N. and Nagata, S. (1993) A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen, J. Biol. Chem., 268, 10932–10937. Yonehara, S., Ishii, A. and Yonehara, M. (1989) A cell-kill-
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ing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor, J. Exp. Med., 169, 1747–1756. Dhein, J., Daniel, P.T., Trauth, B.C., Oehm, A., Mo¨ller, P. and Krammer, P.H. (1992) Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface antigens, J. Immunol., 149, 3166–3173. Banner, D.W., D’Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.-J., Broger, C., Loetscher, H. and Lesslauer, W. (1993) Crystal structure of the soluble human 55 kD TNF receptor-human TNFb complex: implications for TNF receptor activation, Cell, 73, 431–445. Song, H.Y., Dunbar, J.D. and Donner, D.B. (1994) Aggregation of the intracellular domain of the type 1 tumor necrosis factor receptor defined by the two-hybrid system, J. Biol. Chem., 269, 22492–22495. Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schu¨tz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P. and Evans, R.M. (1995) The nuclear receptor superfamily: the second decade, Cell, 83, 835–839.
Expression of Fas-estrogen receptor fusion protein induces cell death in pancreatic cancer cell lines Yoshiya Kawaguchi a,*, Hirohide Takebayashi b, Akira Kakizuka b, Shigeki Arii a, Masayuki Kato a, Masayuki Imamura a a
Department of Surgery and Surgical Basic Science, Kyoto University, 54 Shogoin-Kawaracho, Sakyoku, Kyoto 606, Japan Department of Pharmacology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyoku, Kyoto 606, Japan
b
Received 28 November 1996; revision received 10 February 1997; accepted 21 February 1997
Abstract Recently, a novel system to induce apoptosis was reported. Fusion protein between Fas and the ligand-binding domain of the estrogen receptor (MfasER) could cause apoptotic cell death in an estrogen-dependent manner on murine fibrosarcoma L929 cells and human cervical carcinoma HeLa cells [1]. To investigate the application of this system to the gene therapy of pancreatic cancer, we introduced MfasER cDNA to six cell lines. Transiently expressed MfasER could cause cell death in all the cell lines tested. Furthermore, stably MfasER-expressing cells showed DNA fragmentation as early as 2 h and completely died in 48 h in the presence of estrogen. Combined with effective methods to introduce genes to pancreatic cancer selectively, MfasER would be a good tool for the gene therapy of pancreatic cancer in the future. 1997 Elsevier Science Ireland Ltd. Keywords: Fas; Estrogen receptor; Fusion protein; Gene therapy; Pancreatic cancer
1. Introduction Pancreatic cancer is one of the most difficult diseases to be cured. The majority of this disease is invasive ductal adenocarcinomas that expand very rapidly and form metastatic lesions. Despite the intensive therapies including surgical operation, chemotherapy or radiation therapy, clinicians are faced with disappointing outcomes in most cases [2–5]. The breakthrough to this difficult disease has long been waited for but not seen yet. One possible approach is gene therapy, introducing * Corresponding author.
exogenous genes involved in cell death machinery. Among them, Fas is a type I membrane protein which mediates apoptotic death signal [6]. Recently, a new method to induce apoptosis was reported. Fas fusion protein with the ligand binding domain of the estrogen receptor (MfasER) stably expressed in murine fibrosarcoma L929 cells or human cervical carcinoma HeLa cells could cause apoptotic cell death in an estrogen-dependent manner [1]. In this report, to investigate the application to the gene therapy of pancreatic cancer, we examined the effect of MfasER on six cell lines from human pancreatic cancers and demonstrate that MfasER can induce cell death in all the cell lines tested.
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved P II S0304- 3835 (97 )0 4751- 4
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Y. Kawaguchi et al. / Cancer Letters 116 (1997) 53–59
2. Materials and methods 2.1. Plasmids Construction of MfasER cDNA is almost the same as previously reported [1]. Briefly, cDNA for the transmembrane and intracellular domain of mouse Fas (amino acids 135–305), and cDNA for the ligand binding domain of rat estrogen receptor (amino acids 286–600) are ligated into the expression vector pEF-BOS-bsr with translation initiation site followed by the influenza virus hemagglutinin (HA) epitope, which is recognized by a monoclonal antibody, 12CA5 (Boehringer Mannheim). HA epitope is added at the C-terminal of the fusion protein. Resulting plasmid was named pEF-BOS-bsrMfasER. 2.2. Cell culture Human pancreatic adenocarcinoma cell lines KMP2, 3, 4 and 5 were independently established from resected specimens operated in Kyoto University Hospital (manuscript in preparation; established by Drs. M. K. and Y. Shimada, Kyoto University). MIAPaCa2 and Panc1 were supplied by A.T.C.C. All cell lines were maintained in DMEM supplemented with 10% bovine calf serum (HyClone). 2.3. Transient expression of MfasER and luciferase assay All transfections were carried out with six samples and at least three independent experiments were performed to confirm the reproducibility. Transfections were performed with LipofectAMINE (Gibco BRL) [7]. Cells (2 × 105) were plated on six-well plates and cultured until 50% confluent in DMEM supplemented with 10% serum. Culture medium was removed and cells were washed twice with optiMEM (Gibco BRL). Cells were cotransfected with 1 mg of pEF-BOS-bsr-MfasER and 0.2 mg of pCMXLUC, or 1 mg of pEF-BOS-b-gal and 0.2 mg of pCMX-LUC (as control). After 4 h, transfection medium was replaced with DMEM supplemented with 10% serum, and 12 h later, estradiol (E2) was added at the concentration of 10−9 M. After 33 h of E2 treatment, cells were washed with PBS and har-
vested with cell scraper in 1 ml of PBS. Cells were collected by the centrifugation (15K, 15 s), and resuspended in 100 ml of 0.25 M Tris–HCl (pH 7.5) with Vortex mixer for 10 min. Proteins were eluted by three cycles of freeze-thaw method; each cycle consisted of 2 min in dry ice-cold ethanol and 2 min in water bath at 37°C. After the centrifugation (15K, 10 min), supernatants were collected. Luciferase activities were measured for 30 s with Lumat LB 9501 (Berthold, Germany) as described [7,8]. 2.4. Establishment of stably MfasER-expressing clones The stably MfasER-expressing cells were established as follows: KMP4 and MIAPaCa2 cells were plated on 9 cm dishes, cultured until 50% confluent and transfected with the linearized MfasER expression plasmid by lipofection method. Cells were detached from plates with trypsin and divided into three 9 cm dishes. After selection with blasticidin S (Funakoshi, Japan) at the concentration of 10 mg/ml for 3 weeks, the cells were cloned. 2.5. Assay for cell death Cells were photographed through a Nikon microscope at × 100, 24 h after E2 addition at 10−8 M. For quantification, cells cultured in 96-well plates were stained with crystal violet before and 6, 18, 24, 48 h after the addition of 10−8 M E2. The percentage of viable cells was calculated as described [6,9]. Low molecular weight DNA was purified as follows. Each cell line, plated on 15 cm dish plate, was cultured with or without E2 at 10−8 M for 2 h. Cells were harvested with cell scraper and centrifuged at 1K for 5 min. Collected cell pellets were suspended in 1 ml of PBS and transferred to 1.5 ml tube. After the centrifugation (2K, 5 min), cells were resuspended in 500 ml of 10 mM Tris–HCl (pH 7.5), 10 mM EDTA and 0.2% Triton X-100. Centrifuged at 9K for 10 min at 4°C, the upper aqueous phase was collected, underwent phenol extraction once and phenol-chloroform extraction once, and precipitated with ethanol. DNA pellets were washed with 70% ethanol and suspended in TE buffer. DNA samples were electrophoresed in 2% agarose gels, visualized by ethidium bromide staining and photographed.
Y. Kawaguchi et al. / Cancer Letters 116 (1997) 53–59
Fig. 1. Schematic structure of the Fas, estrogen receptor (ER), and Fas-estrogen receptor fusion protein (MfasER) [1]. The amino acid numbers of the original Fas and ER are shown above and below the schematics, respectively. S, signal peptide; TM, transmembrane domain; DD, death domain; HA, hemagglutinin epitope; LBD, ligand (estradiol) binding domain; DNA, DNA-binding domain.
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Vortex mixer for 15 min and boiled for 5 min. Cell lysates from 5 × 105 cells of parental KMP4 and transiently transfected samples, and 1.7 × 105, 1.7 × 104, 5.7 × 103, 1.9 × 103, 6.3 × 102 of KMP4-34 cells were separated on 10% SDS-polyacrylamide gel and transferred to PVDF membrane. The blot was reacted with the anti-HA monoclonal antibody, 12CA5 (Boehringer Mannheim) [1,7], and the expressed MfasER protein was detected by the alkaline phosphatase color detection system (Promega) for 30 min.
3. Results
2.6. Western blotting Parental KMP4 cells, stably MfasER-expressing KMP4-34 cells, and transiently MfasER-transfected KMP4 cells were harvested and the cell number counted. In transient transfection, samples were obtained 16, 22, and 49 h after transfection. Transfection procedure was the same as above (Section 2.3). At the same time, transfection efficiency was evaluated by X-gal staining of the control transfection. Cells were lysed in 2 × SDS gel-loading buffer with
Schematic structure of MfasER is shown in Fig. 1. The carboxyl-terminal portion of Fas is fused with the ligand binding domain of the estrogen receptor. The transmembrane and death domains of Fas are reported to be essential for mediating apoptotic signal of MfasER [1]. To investigate the cell death effect of MfasER quantitatively, we underwent cotransfection of MfasER expression vector and luciferase expression vector (MfasER+ Luc) to six pancreatic cancer cell lines;
Fig. 2. Relative luciferase activity on transient coexpression of MfasER and Luc (lanes 1 and 2) or b-gal and Luc (lanes 3 and 4 as controls) in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of E2. Relative luciferase activity represents the relative viability of transfected cells (details in the text). From the representative experiment, mean values and standard deviations are shown (n = 6 in each transfection).
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Fig. 3. Estrogen-induced cell death in stably MfasER-expressing cells ( × 73). Cells are healthy in normal medium (B, KMP4-34; D, MIA351), but become round-shaped and detaching from the dishes 24 h after E2 addition (A, KMP4-34; C, MIA35-1).
it was known that the luciferase activities parallel the number of live cells expressing the transfected cDNAs [7]. Luciferase activities of MfasER+ Luc cotransfectant were compared to that of b-gal+ Luc cotransfectant, since b-galactosidase is widely used and believed to show no toxicity. As shown in Fig. 2, transient expression of MfasER induced significant decrease in luciferase activity in the presence of 10−9 M E2 (lane 1), demonstrating that transiently expressed MfasER could cause cell death in all the cell lines tested in the presence of estradiol. This effect was not due to the cell toxicity of estradiol, since the luciferase activities were not influenced by the addition of E2 in control b-gal transfections (lanes 3 and 4). Furthermore, in all the cell lines except MIAPaCa2, decreased luciferase activity was observed even in the absence of E2 (lane 2). This suggests that expressed MfasER has its own constitutive activity (see Section 4). For further investigation, we obtained cell clones that stably express MfasER. Among them, KMP4-34 established from KMP4 and MIA35-1 from MIAPaCa2 were selected. Parental KMP4 and MIAPaCa2 cells showed no morphological changes by the addi-
tion of E2 (data not shown). As shown in Fig. 3, stably MfasER-expressing clones KMP4-34 and MIA35-1 demonstrated clear phenotypic changes in the pre-
Fig. 4. Quantitative analysis of cell viability, evaluated by the crystal violet assay. In the presence of E2, stably MfasER-expressing cells die completely in 48 h.
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Fig. 5. Agarose gel electrophoresis of low molecular weight DNA. Stably MfasER-expressing KMP4-34 and MIA35-1 cells show clear DNA fragmentation 2 h after E2 addition (lanes 1 and 2, respectively), but not in the absence of E2 (lanes 3 and 4, respectively). On the other hand, parental KMP4 and MIAPaCa2 cells do not show DNA fragmentation in the presence (lanes 5 and 6, respectively) nor in the absence (lanes 7 and 8, respectively) of E2.
sence of E2 (Fig. 3A,C, respectively), whereas each clone was healthy in the absence of E2 (Fig. 3B,D, respectively). The cells became round-shaped and shrunken, detaching from the culture plates. These morphological changes became apparent around 18– 24 h treatment of E2, and almost all cells were dead after 48 h. The cell viabilities were quantified by crystal violet assay and are shown in Fig. 4. In the absence of E2, there was no apparent difference in growth rates between parental cells and stable clones (data not shown), but in the presence of E2, stably MfasERexpressing cells died completely in 48 h. Furthermore, DNA fragmentation, a well known marker for apoptosis, was observed as early as 2 h after the addition of E2 in KMP4-34 and MIA35-1 cells (Fig. 5, lanes 1 and 2, respectively), but not apparent in the absence of E2 (Fig. 5, lanes 3 and 4, respectively). On the other hand, parental KMP-4 and MIAPaCa2 cells did not show clear DNA fragmentation regardless of E2 addition (Fig. 5, lanes 5, 6, 7 and 8).
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To evaluate the expression level of MfasER in transiently transfected cells and that in stably expressing cells, we undertook Western blotting using anti-HA monoclonal antibody, 12CA5 [1,7]. As shown in Fig. 6, expressed MfasER protein was detected in the cell lysates from 5 × 105 of transiently transfected cells harvested 16 and 22 h after transfection (lanes 7 and 8, respectively),with the stronger signal than that from 1.7 × 104 of stably-expressing KMP4-34 cells (lane 3). Considering that the transfection efficiency was 0.03%, evaluated by the X-gal-positive cells, 1.5 × 102 cells were expected to express MfasER in 5 × 105 of transiently transfected cells. Thus, compared to the stable clone, more than 100 times the amount of MfasER protein per cell is estimated to be expressed in transient experiment. Forty-nine hours after transfection, the amount of MfasER was decreased in the absence of estradiol (lane 9), indicating the cell death of the MfasER-overexpressing cells. In the presence of estradiol, MfasER became undetectable as early as 6 h after E2 addition (22 h after transfection), or 49 h after transfection (lanes 10 and 11, respectively). It means that estradiol promotes the cell death effect of over-expressed MfasER.
Fig. 6. Detection of expressed MfasER by Western blotting. 5 × 105 of parental KMP4 cells (lane 1), 1.7 × 105, 1.7 × 104, 5.7 × 103, 1.9 × 103 and 6.3 × 102 of stably MfasER-expressing KMP4-34 cells (lanes 2, 3, 4, 5 and 6, respectively), and 5 × 105 of transiently MfasER-transfected KMP4 cells (lanes 7, 8, 9, 10 and 11). In transiently transfected cells, cells were harvested 16, 22 and 49 h after transfection in the absence of estradiol (lanes 7, 8 and 9, respectively), or estradiol was added at 16 h after transfection then harvested 22 and 49 h after transfection (lanes 10 and 11, respectively). Expressed MfasER protein is indicated by the arrow. Note that MfasER is detected from as little as 6.3 × 102 of stably MfasER-expressing KMP4-34 cells (lane 6). Since the transfection efficiency was 0.03%, as counted by the X-gal-positive cells, 1.5 × 102 cells were expected to express MfasER in 5 × 105 of transiently transfected cells.
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4. Discussion In this report, we demonstrated that MfasER could cause cell death in all the pancreatic carcinoma cell lines tested. While there is little information about the expression of Fas in pancreatic cancers, anti-Fas stimulatory antibody CH11 [10] causes cell death in KMP5 and MIAPaCa2 but has little influence on KMP2, 3, 4, or Panc1 (our preliminary observations), suggesting that there is a heterogeneity in Fas-ligandmediated apoptosis among pancreatic cancer cell lines. Nonetheless, as demonstrated here, it should be noted that exogenously introduced MfasER could induce cell death even in the anti-Fas refractory cell lines, indicating that Fas-mediated intracellular signalling pathway is conserved in pancreatic cancer cell lines. Transiently expressed MfasER showed its constitutive activity in all the cell lines except MIAPaCa2, and cell death was promoted in the presence of estradiol (Fig. 2). It has been shown that ligand-dependent formation of oligomeric structure is important in the signal transduction of Fas and TNFR p55 [11,12]. Considering that death domains can multimerize by themselves [13], over-expressed MfasER might form oligomeric structure and transduce the death signal to its target molecules. Furthermore, there is a possibility that the addition of estradiol might facilitate the multimerization, since ER has been shown to form homodimers on its response elements [14]. Of course, the expression level of negative regulators such as bcl2 might possibly differ from cell lines to cell lines. It may account for why the constitutive activities of MfasER were different among cell lines and not apparent in MIAPaCa2. We also demonstrated that stably MfasER-expressing cell lines, KMP4-34 and MIA35-1, showed DNA fragmentation, a well known marker for apoptosis, as early as 2 h after E2 addition. It was far earlier than their phenotypic change. This result was consistent with the previous report [1]. The apoptotic features of estrogen-induced cell death were fully demonstrated in cloned MfasER-expressing L929 cells [1]: DNA fragmentation as early as 1 h after the addition of E2, apparent condensed nuclei by DAPI staining, and typical electron micrograph findings. The IC50 of estradiol was 3 × 10−10 M [1], capable of clinical use without severe side effects. Though we did not per-
form further confirmation in this report, it is easily imagined that MfasER-induced cell death in pancreatic cancer cell lines is also apoptosis. As demonstrated in the Western blotting, more than 100 times the amount of MfasER protein per cell is estimated to be expressed in the transient experiment than that in stable clone and causes constitutive activity in KMP4 cells. For the application to cancer gene therapy, if the MfasER protein is expressed in a cancer-specific manner, this constitutive activity is not a disadvantage. In other words, even the low expression of MfasER, that is, at least 1:100 amount of conventional transfection is capable of causing cell death in the presence of estradiol. It also supports the potential usefulness of MfasER in the gene therapy of pancreatic cancer. In conclusion, as we demonstrated here, MfasER could cause cell death in all the pancreatic carcinoma cell lines tested and its effect was strengthened by the addition of estrogen. For the practical application to the cancer gene therapy, however, many big problems remain to be solved: specific promoter must be cloned, effective methods to deliver exogenous genes must be developed and so on. Unfortunately, there is no effective method to selectively introduce genes to pancreatic cancer to date. Overcoming these obstacles, MfasER would be a good tool for the gene therapy of pancreatic cancer in the future.
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