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Transfecting Well-Differentiated Prostatic Cancer Cell Line LNCaP
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
218, 794–796 (1996)
0141
Transfecting Well-Differentiated Prostatic Cancer Cell Line LNCaP Minna Ruokonen, Jing-Dong Shan, Pirjo Hedberg, Lila Patrikainen, and Pirkko Vihko1 Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland Received November 30, 1995 The transfection of well-differentiated sensitive cells requires careful optimization of the conditions used. In addition to the transfection method chosen, the amount of cells plated, and thus the density of the cells, as well as the influence of the serum concentration play a critical role in the case of LNCaP cells. We also found out that the only appropriate control plasmid for the transfection efficiency was pCMVb-gal driven by the cytomegalovirus promoter. © 1996 Academic Press, Inc.
Despite the abundance of information concerning tissue- and cell-specific transcriptional regulation of many genes, very little is known about the prostate-specific activation of genes. Some information has been gathered with in vitro techniques, e.g. gel retardation and footprinting assays with prostatic nuclear extracts, as well as with mRNA and protein quantifications after different treatments of the cells. The expression of many prostate-specific proteins, e.g. prostate-specific glandular kallikrein, prostate-specific antigen and prostatic acid phosphatase, requires a functionally differentiated status of the cells, androgen-responsiveness and probably the presence of the Y chromosome. In fact, the only established prostatic epithelial cell line available today fulfilling these requirements is LNCaP (1,2). The LNCaP cells originate from a lymph node metastatic lesion of human prostatic adenocarcinoma. The androgen receptor gene of these cells contains a mutation (3), which makes the androgen receptors responsive also to other steroid hormones in addition to androgens. The relatively high degree of differentiation makes LNCaP cells difficult to maintain in cell culture conditions. The low ability of these cells to attach to the growth support, the slow growth rate, the demand for a high percentage of serum in the growth medium, the tendency of making colonies and the modest uptake of DNA are features typical of such differentiated cells. Because of the slow reattachment (60–70%) of dissociated cells, the cell loss during medium changes and the spontaneous detachment of the cells, there exists after several trypsinizations a possibility of random selection which affects the properties of the cell line if it’s heterogeneity is lost. The ability to utilize and maintain well diffentiated cells for transient transfections requires careful optimization of the conditions used. MATERIALS AND METHODS Cell culture and transfections. LNCaP cells were obtained from ATCC, the passages 6–24 were used. The cells were plated at a density of 1 × 106/100 mm plate 68 hours before transfection. Four hours prior to transfection the medium was replaced with 4 ml of medium. The growth medium used before and during the transfection was 10 % FCS-RPMI 1640 (supplemented with L-glutamine, penicillin/streptomycin, fungizone). The transient transfection was performed with lipofectin DOTAP (Boehringer Mannheim). 20 ml (20 mg) DOTAP + 180 ml Hepes (20 mM) and 10 mg DNA + 200 ml Hepes were mixed in separate tubes. The mixes were combined and incubated for at least 10 min before adding to the plates. The total amount of DNA/plate was always 10 mg (3 mg control plasmid pCMVb-gal and 7 mg reporter plasmid pMMTV or pCAT). Mock transfections were included in every series. 24 hours after the beginning of the transfection the medium was
1
Corresponding author. Fax: 358-81-3154451. Abbreviations: CAT, chloramphenicol acetyl transferase; CMV, cytomegalovirus; LNCaP, lymph node carcinoma of the prostate; MMTV, mouse mammary tumor virus; RSV, Rous sarcoma virus; R1881, 17b-Hydroxy-17a-methyl-estra-4,9,11trien-3-one; SV, simian virus. 794 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Activity of the promoterless control vector pCAT and pMMTV in LNCaP cells with and without 10 nM synthetic androgen R1881. replaced with 5% DCC-RPMI with or without androgen (10 nM R1881) and the cells were further incubated for 48 hours. The cell lysates were prepared as usual and the CAT activies were determined as described in (4,5) and the b-galactosidase activities as in (6). 5 ml (1/20) of the lysate was used for b-galactosidase and 30 ml (1/3) for CAT assay. The results presented are mean values of five separate experiments.
RESULTS AND DISCUSSION The density of the cells at the moment of plating is important for successful transfection. If the density is too high, the cells tend to form clumps and detach from the plates. There is also a certain minimum density requirement for LNCaP cells to be able to grow and divide. The density and growth of LNCaP cells have also been shown be be important for the expression and activity of proteins (7). Of the several densities tested (0.5–2 × 106), we found 1 × 10 6 of cells per plate to be optimal. Enough cells and therefore protein was present in the lysates for measurements, but no detachment was observed, although the cells were grown for six days before lysis. Lipofection as a method was chosen because it is the most gentle method for transfection. The activities of pMMTV and the promoterless control vector pCAT in LNCaP cells are shown in Figure 1. The activity of pMMTV is induced 50-fold with 10 nM androgen. We have also transfected LNCaP cells with the DEAE dextran method but the multiple washes and the obvious cytotoxicity of DEAE for LNCaP cells destroyed most of the cells. The calcium phosphate precipitation method can not be used because of the formation of a precipitate with RPMI medium. The concentration of serum in the medium was kept at 10% before and during the transfection to increase the growth rate and to facilitate the uptake of DNA. After the transfection, the serum concentration was again lowered to 5% to enforce the protein synthesis. During the transfection, the amount of medium was minimized to only 4 ml (instead of 10 ml) which was enough for the cells for 24 hours, but there was also a remarkable reduction in the consumption of the transfection reagent needed. The effect of different steroid concentrations on the proliferation of LNCaP cells and the biosynthesis of prostatic proteins has been discussed in (8,9). We have tested in LNCaP cells three control plasmids producing b-galactosidase driven by different promoters. Of the plasmids tested, only the cytomegalovirus promoter containing plasmid (pCMVb-gal) was efficient enough to be considered reliable and reproducible, the pSVb-gal and the pRSVb-gal were only modestly active in LNCaP cells. These results were also confirmed by in situ staining of the cells (data not shown). ACKNOWLEDGMENTS We thank Mrs. Mirja Mäkeläinen, Mrs. Pirkko Ruokojärvi and Mrs. Airi Vesala for expert technical assistance. This work was supported by the Research Council for Medicine of the Academy of Finland. The Department of Clinical Chemistry, 795
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
University of Oulu, is a WHO Collaborating Center for research in reproduction supported by the Ministries of Education, Health and Social Affairs, and Foreign Affairs, Finland.
REFERENCES 1. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., Rosenthal, H., Chu, T. M., Mirand, E. A., and Murphy, G. P. (1983) Cancer Res. 43, 1809–1818. 2. van Steenbrugge, G. J., Groen, M., van Dongen, J. W., Bolt, J., van der Korput, H., Trapman, J., Hasenson, M., and Horoszewicz, J. (1989) Urol. Res. 17, 71–77. 3. Veldscholte, J., Ris-Stalpers, C., Kuiper, G. G. J. M., Jenster, G., Berrevoets, C., Claassen, E., van Rooij, H. C. J., Trapman, J., Brinkmann, A. O., and Mulder, E. (1990) Biochem. Biophys. Res. Comm. 173, 534–540. 4. Neumann, J. R., Morency, C. A., and Russian, K. O. (1987) Biotechniques 5, 444–447. 5. Eastmann, A. (1987) Biotechniques 5, 731. 6. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY. 7. Lin, M. F., DaVolio, J., and Garcia-Arenas, R. (1992) Cancer Res. 52, 4600–4607. 8. Henttu, P. and Vihko, P. (1992) J. Steroid Biochem. Molec. Biol. 41, 349–360. 9. Henttu, P., Liao, S., and Vihko, P. (1992) Endocrinology 130, 766–772.
796
218, 794–796 (1996)
0141
Transfecting Well-Differentiated Prostatic Cancer Cell Line LNCaP Minna Ruokonen, Jing-Dong Shan, Pirjo Hedberg, Lila Patrikainen, and Pirkko Vihko1 Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland Received November 30, 1995 The transfection of well-differentiated sensitive cells requires careful optimization of the conditions used. In addition to the transfection method chosen, the amount of cells plated, and thus the density of the cells, as well as the influence of the serum concentration play a critical role in the case of LNCaP cells. We also found out that the only appropriate control plasmid for the transfection efficiency was pCMVb-gal driven by the cytomegalovirus promoter. © 1996 Academic Press, Inc.
Despite the abundance of information concerning tissue- and cell-specific transcriptional regulation of many genes, very little is known about the prostate-specific activation of genes. Some information has been gathered with in vitro techniques, e.g. gel retardation and footprinting assays with prostatic nuclear extracts, as well as with mRNA and protein quantifications after different treatments of the cells. The expression of many prostate-specific proteins, e.g. prostate-specific glandular kallikrein, prostate-specific antigen and prostatic acid phosphatase, requires a functionally differentiated status of the cells, androgen-responsiveness and probably the presence of the Y chromosome. In fact, the only established prostatic epithelial cell line available today fulfilling these requirements is LNCaP (1,2). The LNCaP cells originate from a lymph node metastatic lesion of human prostatic adenocarcinoma. The androgen receptor gene of these cells contains a mutation (3), which makes the androgen receptors responsive also to other steroid hormones in addition to androgens. The relatively high degree of differentiation makes LNCaP cells difficult to maintain in cell culture conditions. The low ability of these cells to attach to the growth support, the slow growth rate, the demand for a high percentage of serum in the growth medium, the tendency of making colonies and the modest uptake of DNA are features typical of such differentiated cells. Because of the slow reattachment (60–70%) of dissociated cells, the cell loss during medium changes and the spontaneous detachment of the cells, there exists after several trypsinizations a possibility of random selection which affects the properties of the cell line if it’s heterogeneity is lost. The ability to utilize and maintain well diffentiated cells for transient transfections requires careful optimization of the conditions used. MATERIALS AND METHODS Cell culture and transfections. LNCaP cells were obtained from ATCC, the passages 6–24 were used. The cells were plated at a density of 1 × 106/100 mm plate 68 hours before transfection. Four hours prior to transfection the medium was replaced with 4 ml of medium. The growth medium used before and during the transfection was 10 % FCS-RPMI 1640 (supplemented with L-glutamine, penicillin/streptomycin, fungizone). The transient transfection was performed with lipofectin DOTAP (Boehringer Mannheim). 20 ml (20 mg) DOTAP + 180 ml Hepes (20 mM) and 10 mg DNA + 200 ml Hepes were mixed in separate tubes. The mixes were combined and incubated for at least 10 min before adding to the plates. The total amount of DNA/plate was always 10 mg (3 mg control plasmid pCMVb-gal and 7 mg reporter plasmid pMMTV or pCAT). Mock transfections were included in every series. 24 hours after the beginning of the transfection the medium was
1
Corresponding author. Fax: 358-81-3154451. Abbreviations: CAT, chloramphenicol acetyl transferase; CMV, cytomegalovirus; LNCaP, lymph node carcinoma of the prostate; MMTV, mouse mammary tumor virus; RSV, Rous sarcoma virus; R1881, 17b-Hydroxy-17a-methyl-estra-4,9,11trien-3-one; SV, simian virus. 794 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Activity of the promoterless control vector pCAT and pMMTV in LNCaP cells with and without 10 nM synthetic androgen R1881. replaced with 5% DCC-RPMI with or without androgen (10 nM R1881) and the cells were further incubated for 48 hours. The cell lysates were prepared as usual and the CAT activies were determined as described in (4,5) and the b-galactosidase activities as in (6). 5 ml (1/20) of the lysate was used for b-galactosidase and 30 ml (1/3) for CAT assay. The results presented are mean values of five separate experiments.
RESULTS AND DISCUSSION The density of the cells at the moment of plating is important for successful transfection. If the density is too high, the cells tend to form clumps and detach from the plates. There is also a certain minimum density requirement for LNCaP cells to be able to grow and divide. The density and growth of LNCaP cells have also been shown be be important for the expression and activity of proteins (7). Of the several densities tested (0.5–2 × 106), we found 1 × 10 6 of cells per plate to be optimal. Enough cells and therefore protein was present in the lysates for measurements, but no detachment was observed, although the cells were grown for six days before lysis. Lipofection as a method was chosen because it is the most gentle method for transfection. The activities of pMMTV and the promoterless control vector pCAT in LNCaP cells are shown in Figure 1. The activity of pMMTV is induced 50-fold with 10 nM androgen. We have also transfected LNCaP cells with the DEAE dextran method but the multiple washes and the obvious cytotoxicity of DEAE for LNCaP cells destroyed most of the cells. The calcium phosphate precipitation method can not be used because of the formation of a precipitate with RPMI medium. The concentration of serum in the medium was kept at 10% before and during the transfection to increase the growth rate and to facilitate the uptake of DNA. After the transfection, the serum concentration was again lowered to 5% to enforce the protein synthesis. During the transfection, the amount of medium was minimized to only 4 ml (instead of 10 ml) which was enough for the cells for 24 hours, but there was also a remarkable reduction in the consumption of the transfection reagent needed. The effect of different steroid concentrations on the proliferation of LNCaP cells and the biosynthesis of prostatic proteins has been discussed in (8,9). We have tested in LNCaP cells three control plasmids producing b-galactosidase driven by different promoters. Of the plasmids tested, only the cytomegalovirus promoter containing plasmid (pCMVb-gal) was efficient enough to be considered reliable and reproducible, the pSVb-gal and the pRSVb-gal were only modestly active in LNCaP cells. These results were also confirmed by in situ staining of the cells (data not shown). ACKNOWLEDGMENTS We thank Mrs. Mirja Mäkeläinen, Mrs. Pirkko Ruokojärvi and Mrs. Airi Vesala for expert technical assistance. This work was supported by the Research Council for Medicine of the Academy of Finland. The Department of Clinical Chemistry, 795
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
University of Oulu, is a WHO Collaborating Center for research in reproduction supported by the Ministries of Education, Health and Social Affairs, and Foreign Affairs, Finland.
REFERENCES 1. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., Rosenthal, H., Chu, T. M., Mirand, E. A., and Murphy, G. P. (1983) Cancer Res. 43, 1809–1818. 2. van Steenbrugge, G. J., Groen, M., van Dongen, J. W., Bolt, J., van der Korput, H., Trapman, J., Hasenson, M., and Horoszewicz, J. (1989) Urol. Res. 17, 71–77. 3. Veldscholte, J., Ris-Stalpers, C., Kuiper, G. G. J. M., Jenster, G., Berrevoets, C., Claassen, E., van Rooij, H. C. J., Trapman, J., Brinkmann, A. O., and Mulder, E. (1990) Biochem. Biophys. Res. Comm. 173, 534–540. 4. Neumann, J. R., Morency, C. A., and Russian, K. O. (1987) Biotechniques 5, 444–447. 5. Eastmann, A. (1987) Biotechniques 5, 731. 6. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY. 7. Lin, M. F., DaVolio, J., and Garcia-Arenas, R. (1992) Cancer Res. 52, 4600–4607. 8. Henttu, P. and Vihko, P. (1992) J. Steroid Biochem. Molec. Biol. 41, 349–360. 9. Henttu, P., Liao, S., and Vihko, P. (1992) Endocrinology 130, 766–772.
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