THROMBOSIS RESEARCH 65; 409-419,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
NON-SPECIFIC EFFECTS OF AQUAMEPHYTON (VITAMIN Kl) ON PROTHROMBIN EXPRESSION IN HUMAN HEPATOBLASTOMA (HEPGB) CELLS
C. Scott Jamison and Sandra J. Friezner Degen Division of Basic Science Research, Children’s Hospital Medical Center and Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio 45229 (Received 1510.1991;
ABSTRACT
accepted in revised form 12.12.1991
by Editor U. Hedner)
In order to determine the effects of vitamin K1 on prothrombin production, we have treated cultures of human hepatoblastoma cells with an aqueous colloidal suspension of vitamin Kl. Dose-response analysis demonstrated increases in secreted prothrombin antigen levels ranging from 3 to 3.7-fold over controls. Time-course analysis demonstrated increases in secreted prothrombin antigen levels over controls up to 6 hours of treatment. Between 6 and 24 hours, secreted prothrombin antigen levels increased at a rate parallel to controls. Vitamin K1 treatment also resulted in a parallel increase in total Prothrombin mRNA size (approximately secreted protein levels. 2.1 kb) and levels (ranging from 390-480 prothrombin mRNA molecules per cell) were determined by Northern and quantitative solution hybridization analysis, respectively, and were unaffected by vitamin K1 treatment. The increases in secreted prothrombin antigen levels most likely result from non-specific effects of vitamin K1 or agents used to emulsify vitamin K1 on protein release from HepG2 cells.
INTRODUCTION Prothrombin is a vitamin K-dependent blood coagulation protein synthesized Keywords:
Prothrombin, vitamin K1 (phylloquinone, phytonadione), aquaMEPHYTON, blood coagulation, HepG2 cell line 409
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in the liver (l-5). Ten glutamic acid residues near the amino terminus of prothrombin are post-translationally modified to gamma-carboxyglutamic acid residues by a liver microsomal carboxylase in the presence of vitamin K (6-11). In addition to these post-translational effects, vitamin K may also affect other aspects of prothrombin synthesis. Two studies have demonstrated that vitamin K1 treatment of human hepatoblastoma (HepG2) (12) and rat hepatoma (H-35) (13) cell cultures results in increases in secreted prothrombin antigen levels. The authors of these studies suggested that vitamin K1, in addition to its effects on the gamma-carboxylation system, may affect transcription or translation of prothrombin (12, 13). The authors of each of these studies used an aqueous colloidal suspension of vitamin Kl-aquaMEPHYTON (Merck, Sharp, and Dohme, West Point, PA). In order to determine if vitamin K1 treatment affected prothrombin mRNA levels, we have treated HepG2 cultures with aquaMEPHYTON and determined secreted prothrombin antigen, total secreted protein, and prothrombin n-RNA levels.
MATERIALS AND METHODS
Cell culture methods Human hepatoblastoma (HepG2; 14, 15) cells were obtained from Dr. J. A. K. Harmony (University of Cincinnati School of Medicine). HepG2 cells were plated at approximately 106 cells per 100 mm culture dish, cultured in Eagle’s Minimal Essential Medium (EMEM, Whittaker, M A. Bioproducts, Walkersville, MD) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD) and incubated at 37°C in humidified air containing 5% CO2. After a &day incubation, cultures were washed three times with phosphate-buffered saline (PBS: 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4,pH 7.4) and EMEM lacking serum was added. AquaMEPHYTON was obtained from Merck, Sharp, and HepG2 cultures were exposed to 25 pg/ml Dohme (West Point, PA). aquaMEPHYTON or water (control) for the time periods described in the RESULTS. Media samples were removed and stored at -70°C cells were washed with PBS, and RNA isolated as described below. Enzvme-linked immunoso bent assav (ELISA) and D otein determination. Prothrombin levels in meia samples were determined rby an ELISA. Sheep serum containing anti-human prothrombin antibodies (Serotec, Oxford, England) was diluted into Tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.4, 200 mM NaCl), added to 96-well microtiter plates (Elkay Products, Inc., Shrewsbury, MA), and allowed to bind at 4°C overnight. Plates were washed with TBS three times and BSATBS was added as a blocking agent (BSA/TBS: 3% (w/v) bovine serum albumin (Sigma, St. Louis, MO) in TBS). Microtiter plates were incubated at room temperature for 30 minutes. Excess BSA/TBS was removed and samples of HepG2 culture media (100 ~1) were added to wells. Microtiter plates were incubated at room temperature for 1 hour while shaking on a rotator platform. Plates were washed three times with TBS, rabbit serum containing anti-human prothrombin antibody (Nordic Immunologicals, Capistrano Beach, CA) diluted into BSmBS was added, and plates were incubated 1 hours at room temperature while shaking. Plates were washed three times with TBS and goat anti-rabbit IgG peroxidase-conjugated antibody (Boehringer-Mannheim, Indianapolis, IN)
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diluted into BSA/TBS was added. Microtiter plates were incubated with shaking for 1 hour at room temperature. Plates were washed with TBS three times before adding substrate and buffer (17 mM sodium citrate, 65 mM Na2HP04, 45 mM Tris-HCl, 180 mM NaCl, 0.0375% H202, 1 mg/ml o-phenylenediamine, final concentrations). Color evolution was stopped by adding 50% sulfuric acid, and the absorbance at 490 nm was determined. As a standard, human prothrombin (Sigma, St. Louis, MO) was used. Total protein was determined by a Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA). Prothrombin mRNA analvsis. RNA was isolated from control and aquaMEPHYTON-treated HepG2 cultures following the method of Glisin et al. (16). HepG2 cultures were washed three times with ice-cold PBS and cells were lysed by adding a solution containing 1% SDS, 100 kg/ml proteinase K (Sigma, St. Louis, MO), 5 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl, pH 7.5. Cell lysates were incubated at 45°C for 2 hours, followed by extraction with phenol and chloroform. Cesium chloride was added to the aqueous layer to a final concentration of 1 g/ml. Total RNA was collected as a pellet under a CsCl cushion (0.96 g/ml CsCl, final concentration), by centrifugation in an SW-60 rotor (Beckman, Fullerton, CA) at 35,000 rpm for approximately 16 hours. Northern analysis has been previously described (17). Samples of total RNA (2Opg) were subjected to electrophoresis in 1% agarose gels containing 2.4 M formaldehyde. Ethidium bromide (1 pg per sample) was added prior to electrophoresis to allow visualization of the RNA with ultraviolet light and ensure that equivalent amounts of RNA were present in each lane. After electrophoresis, RNA was transferred to Biotrans membrane (ICN Biochemicals, Costa Mesa, CA). A 1683 bp XhoI-EcoRI fragment of a human prothrombin cDNA (18) was randomprimer labelled (19,20) with [a- 32PldCTP (DuPont/New England Nuclear, Boston, MA). Hybridization conditions were 60°C overnight with 106 cpm of 32P-labelled probe per milliliter of hybidization solution. The membrane was exposed to X-ray film (X-OMAT AR, Eastman Kodak) for 36 hours with an intensifying screen at -70°C. For quantitation of prothrombin mRNA, solution hybridization analysis was performed as described previously (21). In brief, a 30 base oligonucleotide was 5’ end-labelled with [y- 32PldATP and T4 polynucleotide kinase and subsequently allowed to hybridize with samples of total RNA or a single-stranded standard DNA. Excess labelled oligonucleotide was digested with Sl nuclease and stable hybrids were precipitated with trichloroacetic acid. Precipitates were collected on glass fiber filters (Whatman, GFC) and Sl-resistant cpm were determined by scintillation spectroscopy. The quantity of prothrombin mRNA molecules per cell were determined by comparison of cpm hybridized to total RNA samples with cpm hybridized to the standard DNA. The number of prothrombin mRNA molecules per cell were calculated from size of the standard DNA (9250 nucleotides) and the quantity of total RNA per cell (9.4 ug total RNA per 106 HepG2 cells, determined in this laboratory) as described (21). RESULTS Time-course for secretion of prothrombin antipen. It is apparent from previous studies that treatment of cultured cells with aquaMEPHYTON results in an increase in secreted prothrombin antigen levels (12, 13). This increase only
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occurs during the initial 24 hours of treatment. After 24 hours, secreted prothrombin levels in media samples increase at a parallel rate to controls (12, 13). In order to determine the time-course of effects of AquaMEPHYTON on secreted prothrombin antigen levels in our system, HepG2 cells in serum-free media were treated with 25 pg/ml aquaMEPHYTON and levels of secreted prothrombin antigen were determined by an ELISA (Figure 1).
600 500 400 300 200 100 0 5
10
15
20
25
Time (h)
Figure 1. Time-course of prothrombin antigen secreted by HepG2 cultures after Secreted prothrombin antigen levels were aquaMEPHYTON treatment. determined in media samples from cultures of HepG2 cells treated with 25 pg/ml aquaMEPHYTON (0) or water (control) (0). Prothrombin antigen levels were determined by an ELBA and quantitated by using human prothrombin (Sigma, St. Louis, MO) as a standard. The number of determinations at each timepoint (1.5, 3, 6, and 24 hours) were 5, 8, 9, and 9 for control cultures and 8, 10, 9, and 8 for aquaMEPHYTON-treated cultures, respectively. At each timepoint, values for prothrombin antigen were significantly increased with aquaMEPHYTON treatment versus controls (Student’s t-test, p-zO.01)
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i Do$!+.e-;p~e analvsls Of effects Of aauaMEPHYToN On secreted DrothrQUk n order to determine if 25 pg/ml aquaMEPHYTON was an appropriate level to be used in these experiments, a dose-response analysis was performed. HepG2 cultures in serum-free media were treated with various amounts of Media aquaMEPHYTON ranging from 0 to 100 pg/ml final concentration. samples were removed at 6 hours and prothrombin antigen levels determined by an ELISA. Treatment with aquaMEPHYTON for 6 hours resulted in increases in secreted prothrombin antigen levels ranging from 3.0- to 3.7-fold over controls (Table 1). TABLE 1. Dose-Response Analysis of Secreted Prothrombin Antigen Levels to Various Doses of aquaMEPHYTON with 6 Hours of Treatment aquaMEPHYTON concentration (pg/ml)
0
10 25 50 100
Prothrombin antigen concentration (ng/mkkstd. dev.)a
41.9k10.2 131.0&23.3 151.W2.3 154.9k32.6 125.6k30.9
Fold Increase
_____ 3.1 3.6 3.7 3.0
aFor each of the values for prothrombin antigen at 0, 10, 25, 50, and 100 pg/ml aquaMEPHYTON, the number of determinations were 5, 10, 6, 6, and 7, respectively. The levels of nrothrombin antigen from each of the aquaMEPHYTON-treated cultures kere significantly in&eased over control levels (Student’s t-test, pcO.01).
Ti me -cour se of effects of aau,&MEPHYTON on total secreted nrotein levels, In order to determine if the increase in secreted prothrombin antigen was the result of specific effects of aquaMEPHYTON treatment on prothrombin antigen or the result of general effects on total secreted protein levels, total secreted protein levels in media samples were determined. Treatment of HepG2 cultures with aquaMEPHYTON resulted in increases in total secreted protein levels over control levels (Figure 2). levels, If HYTON on secreted nrothre treatment with aquaMEPHYTON results in a specific increase in secreted prothrombin antigen levels, the ratio of secreted prothrombin antigen levels to total secreted protein levels should be greater for aquaMEPHYTON-treated HepG2 cultures than for control cultures. At two timepoints (1.5, 6 hours) the ratio for
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aquaMEPHYTON-treated cultures was slightly greater than that found for control cultures. At the other timepoints, the ratio for aquaMEPHYTON-treated HepG2 cultures was slightly less than the ratio for control samples (Table 2). of nrothrombin mRNA, One possibility for the increase in secreted prothrombin and total protein levels with aquaMEPHYTON treatment could be that mRNA levels for prothrombin and other proteins were increased. Northern and solution hybridization analyses were used to determine if aquaMEPHYTON treatment resulted in an increase in prothrombin mRNA levels. Northern analysis demonstrated that prothrombin mRNA size (approximately 2.1 kb) did not vary with treatment (Figure 3). Solution hybridization analysis demonstrated that the quantity of prothrombin mRNA did not vary with aquaMEPHYTON treatment (Table 3).
Analvsis
5
lo
15
20
25
Time (hour)
Figure 2. Time-course of total protein secreted by HepG2 cultures after aquaMEPHYTON treatment. Secreted total protein levels were quantitated in media samples from cultures of HepG2 cells treated with 25 pg/ml aquaMEPHYTON (0) or water (control) (a). Protein levels were determined from duplicate samples by protein assay using a Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA) and bovine serum albumin (Sigma, St. Louis, MO) as a standard.
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TABLE 2. Levels of Secreted Prothrombin Antigen Relative to Total Secreted Protein Levels. prothrombin (ngYtota1 protein (vg) Time (h)
0 IJglml aquaMEPHYTON
25 kg/ml aquaMEPHYTON
1.5 3 6 24
2.02 3.06 2.55 3.45
2.87 2.23 2.73 3.16
Fold Change
1.42 0.73 1.07 0.92
TABLE 3. Quantitative (Solution Hybridization) Analysis of Prothrombin mRNA. Prothrombin Time (h) 0
1.5 3 6 24
0 pg/ml aquaMEPHYTON 402.5k27.3 448.Zk77.5 389.5rt17.5 428.4k64.7 479.7k55.4
mRNA moleculeskella 25 pg/ml aquaMEPHYTON _____ 409.6k47.0 432.7k47.7 410.7k37.9 432.1k65.5
aAn analysis of variance was performed and the hypothesis that at least one of the values for prothrombin mRNA molecules/cell was not equivalent to the others was rejected (pcO.005).
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123456789 Origin -
28s -
18s -
Figure 3. Northern analysis of total RNA isolated from HepG2 cultures treated with 25 pglml aquaMEPHYTON or water (control). Samples of total RNA were subjected to electrophoresis on 1% agarose gels containing 2.4 M formaldehyde, transferred to a Biotrans membrane (ICN Biochemicals, Costa Mesa, CA) and allowed to hybridize with a 32P-labelled human prothrombin cDNA probe (18). Lane 1 corresponds to total RNA isolated from a control HepG2 culture at time zero. Lanes 2-5 and 6-9 correspond to total RNA isolated from control or aquaMEPHYTON-treated HepG2 cultures, respectively, at 1.5 (lanes 2 and 61, 3 (lanes 3 and 71, 6 (lanes 4 and 8), and 24 (lanes 5 and 9) hours of treatment.
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DISCUSSION Two groups of authors have demonstrated that aquaMEPHYTON treatment of cultured cells results in increases in secreted prothrombin antigen levels (12, 13). The first group (12) treated HepG2 cultures with 25 yg/ml aquaMEPHYTON for 24 hours and demonstrated a 2-fold increase in secreted prothrombin antigen levels as compared to controls. The second group (13) treated rat hepatoma (H-35) cells with 0.1 pg/ml aquaMEPHYTON for 24 hours and demonstrated an approximately &fold increase in secreted prothrombin levels. These authors suggested that these increases may result from the effects of vitamin K1 on prothrombin mRNA levels (12, 13). Neither of these groups (12, 13) discussed the effect of aquaMEPHYTON treatment on total secreted protein levels. In the course of studies to determine if vitamin K1 treatment affected secreted prothrombin antigen and mRNA levels produced by HepG2 cells, we used two sources of vitamin Kl. One source of vitamin K1 was obtained from Sigma (St. Louis, MO). Treatment of HepG2 cells with this form of vitamin Kl, an oil soluble in ethanol, resulted in an approximately 2.0-fold increase in secreted prothrombin antigen levels with 3 hours of 25 pg/ml vitamin K1 treatment as determined by an ELBA (unpublished results). There was no apparent effect on total secreted protein or prothrombin mRNA levels (unpublished results). The second source of vitamin K1 was aquaMEPHYTON (Merck, Sharp, and Dohme, West Point, PA), an aqueous colloidal suspension of vitamin Kl. After treatment of HepG2 cells with aquaMEPHYTON, we observed increases in secreted prothrombin antigen levels as compared to controls, however, these increases were paralleled by increases in total secreted protein levels as compared to controls. In order to determine if the increase in secreted prothrombin levels was due to an effect at the transcriptional level, we isolated total RNA from aquaMEPHYTON-treated and control cultures and subjected these RNA samples to Northern analysis and a quantitative solution hybridization analysis. There were no detectable differences in either size (approximately 2.1 kb) or levels of prothrombin mRNA between samples. It is apparent from our results that aquaMEPHYTON treatment of HepG2 cells results in non-specific increases in secreted prothrombin levels. These non-specific effects may be due to effects either of vitamin K1 or of agents used to emulsify vitamin K1 in aquaMEPHYTON. Since we have evidence (unpublished results) that treatment of HepG2 cultures with vitamin K1 oil results in specific increases in secreted prothrombin levels, we suggest that the non-specific effects of aquaMEPHYTON treatment result from agents used to emulsify vitamin K1 in this compound. We and previous authors (12, 13) used serum-free media during treatment of cell cultures with aquaMEPHYTON. It will be of interest to determine if the presence of fetal bovine or other sera alters the effects of aquaMEPHYTON on cell lines and perhaps prevents the release of total protein from these cells. It is appropriate to mention that, although this report provides evidence for non-specific effects of aquaMEPHYTON on cells, this compound is apparently nontoxic to humans (22). We were able to find only one report of complications resulting from the administration of aquaMEPHYTON to humans (23). This case report described an association between intravenous administration of aquaMEPHYTON and acute development of hypotension and subsequent cardiovascular collapse (23). The authors suggested that vitamin Kl, or the agents used to emulsify vitamin Kl, may have been responsible for the vasodilation (23).
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ACKNOWLEDGEMENTS This work was supported by a Molecular and Cellular Biology Post-Doctoral Training Grant NIH HL07527 (C.S.J.), an NIH research grant HL38232 (S.J.F.D.) and The Pew Memorial Trust (S.J.F.D.). S.J.F.D. is a Pew Scholar in the Biomedical Sciences and an Established Investigator of the American Heart Association. REFERENCES
1.
Davie, E.W. and Fujikawa, K. Basic mechanisms Annu Rev Biochem 44,799-3291975.
2.
Davie, E.W., Fujikawa, K., Kurachi, K. and Kisiel, W. The role of serine proteases in the blood coagulation cascade. Adv Enzymol48,277-318, 1979.
3.
Jackson, C.M. and Nemerson, Y. Blood coagulation. 765811,198O.
4.
Kisiel, W. and Hanahan, D.J. Purification and characterization factor II. Biochim Biophys Acta 304, 103-113, 1973.
5.
Barnhart, M.I. 360-366,196O.
6.
Walz, D.A., Hewett-Emmett, D., Guillin, M.-C. Amino acid sequences and molecular homology of the vitamin K-dependent clotting factors. In: Prothrombin and Other Vitamin K Proteins, vol 1, W H Seegers and D A Walz (Eds.) Boca Raton, FL: CRC Press, Inc 1986, pp. 125-160.
7.
Stenflo, J., Fernlund, P., Egan, W. and Roepstorff, P. Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad Sci, USA 71,2730-2733,1974.
8.
Nelsestuen, G.L., Zytkovicz, T.H. and Howard, J.B. The mode of action of vitamin K: Identification of gamma-carboxyglutamic acid as a component of prothrombin. J Biol Chem 249,6347-6350,1974.
9.
Stenflo, J. and Suttie, J.W. Vitamin K-dependent formation of gammacarboxyglutamic acid. Annu Rev Biochem 46, 157-172,1977.
Annu Rev Biochem 49,
Cellular site for prothrombin synthesis.
10. Suttie, J.W. Vitamin K-dependent carboxylase. 477,1985. 11.
in blood coagulation.
of human
Am J Physiol 199,
Annu Rev Biochem 54, 459-
Esmon, C.T., Sadowski, J.A. and Suttie, J.W. A new carboxylation reaction: the vitamin K-dependent incorporation of H14CO3- into prothrombin. J Biol Chem 250,4744-4748,1975.
Vol. 65,No.3
419
VlTAMlNK,&PROTHROMBlNmRNALEVELS
12. Fair, D.S. and Bahnak, B.R.
Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III. Blood 64, 194-204, 1984.
13. Munns,
T.W., Johnston, M.F.M., Liszewski, M.K. and Olson, R.E.L. Vitamin K-dependent synthesis and modification of precursor prothrombin in cultured H-35 hepatoma cells. Proc iVutZ Acad Sci, USA 73, 2803-2807, 1976. Human hepatocellular B.B., Howe, C.C. and Aden, D.P. carcinoma cell lines secreted the major plasma proteins and hepatitis B surface antigen. Science 209,497-499, 1980.
14. Knowles,
15. Javitt, N.B. Hep G2 cells as a resource for metabolic studies: cholesterol, and bile acids. FASEB J4, 161-168, 1990.
lipoprotein,
16. Glisin, V., Crkvenjakov, R. and Byus, C. Ribonucleic acid isolated by cesium chloride centrimgation.
Biochemistry 13, 2633-2637, 1974.
17. Jamison,
C.S. and Degen, S.J.F. mRNA coding for rat prothrombin. 1991.
Prenatal and postnatal expression of Biochim Biophys Acta 1088, 208-216,
18. Degen, S.J.F., MacGillivray, R.T.F. and Davie, E.W. Characterization of the complementary deoxyribonucleic acid and prothrombin. Biochemistry 22, 2087-2097, 1983.
gene
coding
for
human
19. Feinberg,
A.P. and Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132,6-13,1983.
20. Feinberg,
A.P. and Vogelstein, B. ADDENDUM: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137,266-267, 1984.
21. Durnam, D.M. and Palmiter, R.D.
A practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem 131, 385-393, 1983.
22. Marcus, R. and Coulston, A.M.
Fat-soluble vitamins: Vitamins A, K, and E. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 8th edition, A G Gilman, T W Rall, A S Nies and P Taylor (Eds.) New York: Pergamon Press 1990, pp. 1563-1566.
23.
Barash, P., Kitahata, L.M. and Mandel, S. Acute cardiovascular collapse after intravenous phytonadione. Anesth Analg Curr Res 55, 304-306, 1976.
NON-SPECIFIC EFFECTS OF AQUAMEPHYTON (VITAMIN Kl) ON PROTHROMBIN EXPRESSION IN HUMAN HEPATOBLASTOMA (HEPGB) CELLS
C. Scott Jamison and Sandra J. Friezner Degen Division of Basic Science Research, Children’s Hospital Medical Center and Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio 45229 (Received 1510.1991;
ABSTRACT
accepted in revised form 12.12.1991
by Editor U. Hedner)
In order to determine the effects of vitamin K1 on prothrombin production, we have treated cultures of human hepatoblastoma cells with an aqueous colloidal suspension of vitamin Kl. Dose-response analysis demonstrated increases in secreted prothrombin antigen levels ranging from 3 to 3.7-fold over controls. Time-course analysis demonstrated increases in secreted prothrombin antigen levels over controls up to 6 hours of treatment. Between 6 and 24 hours, secreted prothrombin antigen levels increased at a rate parallel to controls. Vitamin K1 treatment also resulted in a parallel increase in total Prothrombin mRNA size (approximately secreted protein levels. 2.1 kb) and levels (ranging from 390-480 prothrombin mRNA molecules per cell) were determined by Northern and quantitative solution hybridization analysis, respectively, and were unaffected by vitamin K1 treatment. The increases in secreted prothrombin antigen levels most likely result from non-specific effects of vitamin K1 or agents used to emulsify vitamin K1 on protein release from HepG2 cells.
INTRODUCTION Prothrombin is a vitamin K-dependent blood coagulation protein synthesized Keywords:
Prothrombin, vitamin K1 (phylloquinone, phytonadione), aquaMEPHYTON, blood coagulation, HepG2 cell line 409
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in the liver (l-5). Ten glutamic acid residues near the amino terminus of prothrombin are post-translationally modified to gamma-carboxyglutamic acid residues by a liver microsomal carboxylase in the presence of vitamin K (6-11). In addition to these post-translational effects, vitamin K may also affect other aspects of prothrombin synthesis. Two studies have demonstrated that vitamin K1 treatment of human hepatoblastoma (HepG2) (12) and rat hepatoma (H-35) (13) cell cultures results in increases in secreted prothrombin antigen levels. The authors of these studies suggested that vitamin K1, in addition to its effects on the gamma-carboxylation system, may affect transcription or translation of prothrombin (12, 13). The authors of each of these studies used an aqueous colloidal suspension of vitamin Kl-aquaMEPHYTON (Merck, Sharp, and Dohme, West Point, PA). In order to determine if vitamin K1 treatment affected prothrombin mRNA levels, we have treated HepG2 cultures with aquaMEPHYTON and determined secreted prothrombin antigen, total secreted protein, and prothrombin n-RNA levels.
MATERIALS AND METHODS
Cell culture methods Human hepatoblastoma (HepG2; 14, 15) cells were obtained from Dr. J. A. K. Harmony (University of Cincinnati School of Medicine). HepG2 cells were plated at approximately 106 cells per 100 mm culture dish, cultured in Eagle’s Minimal Essential Medium (EMEM, Whittaker, M A. Bioproducts, Walkersville, MD) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD) and incubated at 37°C in humidified air containing 5% CO2. After a &day incubation, cultures were washed three times with phosphate-buffered saline (PBS: 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4,pH 7.4) and EMEM lacking serum was added. AquaMEPHYTON was obtained from Merck, Sharp, and HepG2 cultures were exposed to 25 pg/ml Dohme (West Point, PA). aquaMEPHYTON or water (control) for the time periods described in the RESULTS. Media samples were removed and stored at -70°C cells were washed with PBS, and RNA isolated as described below. Enzvme-linked immunoso bent assav (ELISA) and D otein determination. Prothrombin levels in meia samples were determined rby an ELISA. Sheep serum containing anti-human prothrombin antibodies (Serotec, Oxford, England) was diluted into Tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.4, 200 mM NaCl), added to 96-well microtiter plates (Elkay Products, Inc., Shrewsbury, MA), and allowed to bind at 4°C overnight. Plates were washed with TBS three times and BSATBS was added as a blocking agent (BSA/TBS: 3% (w/v) bovine serum albumin (Sigma, St. Louis, MO) in TBS). Microtiter plates were incubated at room temperature for 30 minutes. Excess BSA/TBS was removed and samples of HepG2 culture media (100 ~1) were added to wells. Microtiter plates were incubated at room temperature for 1 hour while shaking on a rotator platform. Plates were washed three times with TBS, rabbit serum containing anti-human prothrombin antibody (Nordic Immunologicals, Capistrano Beach, CA) diluted into BSmBS was added, and plates were incubated 1 hours at room temperature while shaking. Plates were washed three times with TBS and goat anti-rabbit IgG peroxidase-conjugated antibody (Boehringer-Mannheim, Indianapolis, IN)
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diluted into BSA/TBS was added. Microtiter plates were incubated with shaking for 1 hour at room temperature. Plates were washed with TBS three times before adding substrate and buffer (17 mM sodium citrate, 65 mM Na2HP04, 45 mM Tris-HCl, 180 mM NaCl, 0.0375% H202, 1 mg/ml o-phenylenediamine, final concentrations). Color evolution was stopped by adding 50% sulfuric acid, and the absorbance at 490 nm was determined. As a standard, human prothrombin (Sigma, St. Louis, MO) was used. Total protein was determined by a Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA). Prothrombin mRNA analvsis. RNA was isolated from control and aquaMEPHYTON-treated HepG2 cultures following the method of Glisin et al. (16). HepG2 cultures were washed three times with ice-cold PBS and cells were lysed by adding a solution containing 1% SDS, 100 kg/ml proteinase K (Sigma, St. Louis, MO), 5 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl, pH 7.5. Cell lysates were incubated at 45°C for 2 hours, followed by extraction with phenol and chloroform. Cesium chloride was added to the aqueous layer to a final concentration of 1 g/ml. Total RNA was collected as a pellet under a CsCl cushion (0.96 g/ml CsCl, final concentration), by centrifugation in an SW-60 rotor (Beckman, Fullerton, CA) at 35,000 rpm for approximately 16 hours. Northern analysis has been previously described (17). Samples of total RNA (2Opg) were subjected to electrophoresis in 1% agarose gels containing 2.4 M formaldehyde. Ethidium bromide (1 pg per sample) was added prior to electrophoresis to allow visualization of the RNA with ultraviolet light and ensure that equivalent amounts of RNA were present in each lane. After electrophoresis, RNA was transferred to Biotrans membrane (ICN Biochemicals, Costa Mesa, CA). A 1683 bp XhoI-EcoRI fragment of a human prothrombin cDNA (18) was randomprimer labelled (19,20) with [a- 32PldCTP (DuPont/New England Nuclear, Boston, MA). Hybridization conditions were 60°C overnight with 106 cpm of 32P-labelled probe per milliliter of hybidization solution. The membrane was exposed to X-ray film (X-OMAT AR, Eastman Kodak) for 36 hours with an intensifying screen at -70°C. For quantitation of prothrombin mRNA, solution hybridization analysis was performed as described previously (21). In brief, a 30 base oligonucleotide was 5’ end-labelled with [y- 32PldATP and T4 polynucleotide kinase and subsequently allowed to hybridize with samples of total RNA or a single-stranded standard DNA. Excess labelled oligonucleotide was digested with Sl nuclease and stable hybrids were precipitated with trichloroacetic acid. Precipitates were collected on glass fiber filters (Whatman, GFC) and Sl-resistant cpm were determined by scintillation spectroscopy. The quantity of prothrombin mRNA molecules per cell were determined by comparison of cpm hybridized to total RNA samples with cpm hybridized to the standard DNA. The number of prothrombin mRNA molecules per cell were calculated from size of the standard DNA (9250 nucleotides) and the quantity of total RNA per cell (9.4 ug total RNA per 106 HepG2 cells, determined in this laboratory) as described (21). RESULTS Time-course for secretion of prothrombin antipen. It is apparent from previous studies that treatment of cultured cells with aquaMEPHYTON results in an increase in secreted prothrombin antigen levels (12, 13). This increase only
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occurs during the initial 24 hours of treatment. After 24 hours, secreted prothrombin levels in media samples increase at a parallel rate to controls (12, 13). In order to determine the time-course of effects of AquaMEPHYTON on secreted prothrombin antigen levels in our system, HepG2 cells in serum-free media were treated with 25 pg/ml aquaMEPHYTON and levels of secreted prothrombin antigen were determined by an ELISA (Figure 1).
600 500 400 300 200 100 0 5
10
15
20
25
Time (h)
Figure 1. Time-course of prothrombin antigen secreted by HepG2 cultures after Secreted prothrombin antigen levels were aquaMEPHYTON treatment. determined in media samples from cultures of HepG2 cells treated with 25 pg/ml aquaMEPHYTON (0) or water (control) (0). Prothrombin antigen levels were determined by an ELBA and quantitated by using human prothrombin (Sigma, St. Louis, MO) as a standard. The number of determinations at each timepoint (1.5, 3, 6, and 24 hours) were 5, 8, 9, and 9 for control cultures and 8, 10, 9, and 8 for aquaMEPHYTON-treated cultures, respectively. At each timepoint, values for prothrombin antigen were significantly increased with aquaMEPHYTON treatment versus controls (Student’s t-test, p-zO.01)
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i Do$!+.e-;p~e analvsls Of effects Of aauaMEPHYToN On secreted DrothrQUk n order to determine if 25 pg/ml aquaMEPHYTON was an appropriate level to be used in these experiments, a dose-response analysis was performed. HepG2 cultures in serum-free media were treated with various amounts of Media aquaMEPHYTON ranging from 0 to 100 pg/ml final concentration. samples were removed at 6 hours and prothrombin antigen levels determined by an ELISA. Treatment with aquaMEPHYTON for 6 hours resulted in increases in secreted prothrombin antigen levels ranging from 3.0- to 3.7-fold over controls (Table 1). TABLE 1. Dose-Response Analysis of Secreted Prothrombin Antigen Levels to Various Doses of aquaMEPHYTON with 6 Hours of Treatment aquaMEPHYTON concentration (pg/ml)
0
10 25 50 100
Prothrombin antigen concentration (ng/mkkstd. dev.)a
41.9k10.2 131.0&23.3 151.W2.3 154.9k32.6 125.6k30.9
Fold Increase
_____ 3.1 3.6 3.7 3.0
aFor each of the values for prothrombin antigen at 0, 10, 25, 50, and 100 pg/ml aquaMEPHYTON, the number of determinations were 5, 10, 6, 6, and 7, respectively. The levels of nrothrombin antigen from each of the aquaMEPHYTON-treated cultures kere significantly in&eased over control levels (Student’s t-test, pcO.01).
Ti me -cour se of effects of aau,&MEPHYTON on total secreted nrotein levels, In order to determine if the increase in secreted prothrombin antigen was the result of specific effects of aquaMEPHYTON treatment on prothrombin antigen or the result of general effects on total secreted protein levels, total secreted protein levels in media samples were determined. Treatment of HepG2 cultures with aquaMEPHYTON resulted in increases in total secreted protein levels over control levels (Figure 2). levels, If HYTON on secreted nrothre treatment with aquaMEPHYTON results in a specific increase in secreted prothrombin antigen levels, the ratio of secreted prothrombin antigen levels to total secreted protein levels should be greater for aquaMEPHYTON-treated HepG2 cultures than for control cultures. At two timepoints (1.5, 6 hours) the ratio for
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aquaMEPHYTON-treated cultures was slightly greater than that found for control cultures. At the other timepoints, the ratio for aquaMEPHYTON-treated HepG2 cultures was slightly less than the ratio for control samples (Table 2). of nrothrombin mRNA, One possibility for the increase in secreted prothrombin and total protein levels with aquaMEPHYTON treatment could be that mRNA levels for prothrombin and other proteins were increased. Northern and solution hybridization analyses were used to determine if aquaMEPHYTON treatment resulted in an increase in prothrombin mRNA levels. Northern analysis demonstrated that prothrombin mRNA size (approximately 2.1 kb) did not vary with treatment (Figure 3). Solution hybridization analysis demonstrated that the quantity of prothrombin mRNA did not vary with aquaMEPHYTON treatment (Table 3).
Analvsis
5
lo
15
20
25
Time (hour)
Figure 2. Time-course of total protein secreted by HepG2 cultures after aquaMEPHYTON treatment. Secreted total protein levels were quantitated in media samples from cultures of HepG2 cells treated with 25 pg/ml aquaMEPHYTON (0) or water (control) (a). Protein levels were determined from duplicate samples by protein assay using a Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA) and bovine serum albumin (Sigma, St. Louis, MO) as a standard.
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TABLE 2. Levels of Secreted Prothrombin Antigen Relative to Total Secreted Protein Levels. prothrombin (ngYtota1 protein (vg) Time (h)
0 IJglml aquaMEPHYTON
25 kg/ml aquaMEPHYTON
1.5 3 6 24
2.02 3.06 2.55 3.45
2.87 2.23 2.73 3.16
Fold Change
1.42 0.73 1.07 0.92
TABLE 3. Quantitative (Solution Hybridization) Analysis of Prothrombin mRNA. Prothrombin Time (h) 0
1.5 3 6 24
0 pg/ml aquaMEPHYTON 402.5k27.3 448.Zk77.5 389.5rt17.5 428.4k64.7 479.7k55.4
mRNA moleculeskella 25 pg/ml aquaMEPHYTON _____ 409.6k47.0 432.7k47.7 410.7k37.9 432.1k65.5
aAn analysis of variance was performed and the hypothesis that at least one of the values for prothrombin mRNA molecules/cell was not equivalent to the others was rejected (pcO.005).
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123456789 Origin -
28s -
18s -
Figure 3. Northern analysis of total RNA isolated from HepG2 cultures treated with 25 pglml aquaMEPHYTON or water (control). Samples of total RNA were subjected to electrophoresis on 1% agarose gels containing 2.4 M formaldehyde, transferred to a Biotrans membrane (ICN Biochemicals, Costa Mesa, CA) and allowed to hybridize with a 32P-labelled human prothrombin cDNA probe (18). Lane 1 corresponds to total RNA isolated from a control HepG2 culture at time zero. Lanes 2-5 and 6-9 correspond to total RNA isolated from control or aquaMEPHYTON-treated HepG2 cultures, respectively, at 1.5 (lanes 2 and 61, 3 (lanes 3 and 71, 6 (lanes 4 and 8), and 24 (lanes 5 and 9) hours of treatment.
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DISCUSSION Two groups of authors have demonstrated that aquaMEPHYTON treatment of cultured cells results in increases in secreted prothrombin antigen levels (12, 13). The first group (12) treated HepG2 cultures with 25 yg/ml aquaMEPHYTON for 24 hours and demonstrated a 2-fold increase in secreted prothrombin antigen levels as compared to controls. The second group (13) treated rat hepatoma (H-35) cells with 0.1 pg/ml aquaMEPHYTON for 24 hours and demonstrated an approximately &fold increase in secreted prothrombin levels. These authors suggested that these increases may result from the effects of vitamin K1 on prothrombin mRNA levels (12, 13). Neither of these groups (12, 13) discussed the effect of aquaMEPHYTON treatment on total secreted protein levels. In the course of studies to determine if vitamin K1 treatment affected secreted prothrombin antigen and mRNA levels produced by HepG2 cells, we used two sources of vitamin Kl. One source of vitamin K1 was obtained from Sigma (St. Louis, MO). Treatment of HepG2 cells with this form of vitamin Kl, an oil soluble in ethanol, resulted in an approximately 2.0-fold increase in secreted prothrombin antigen levels with 3 hours of 25 pg/ml vitamin K1 treatment as determined by an ELBA (unpublished results). There was no apparent effect on total secreted protein or prothrombin mRNA levels (unpublished results). The second source of vitamin K1 was aquaMEPHYTON (Merck, Sharp, and Dohme, West Point, PA), an aqueous colloidal suspension of vitamin Kl. After treatment of HepG2 cells with aquaMEPHYTON, we observed increases in secreted prothrombin antigen levels as compared to controls, however, these increases were paralleled by increases in total secreted protein levels as compared to controls. In order to determine if the increase in secreted prothrombin levels was due to an effect at the transcriptional level, we isolated total RNA from aquaMEPHYTON-treated and control cultures and subjected these RNA samples to Northern analysis and a quantitative solution hybridization analysis. There were no detectable differences in either size (approximately 2.1 kb) or levels of prothrombin mRNA between samples. It is apparent from our results that aquaMEPHYTON treatment of HepG2 cells results in non-specific increases in secreted prothrombin levels. These non-specific effects may be due to effects either of vitamin K1 or of agents used to emulsify vitamin K1 in aquaMEPHYTON. Since we have evidence (unpublished results) that treatment of HepG2 cultures with vitamin K1 oil results in specific increases in secreted prothrombin levels, we suggest that the non-specific effects of aquaMEPHYTON treatment result from agents used to emulsify vitamin K1 in this compound. We and previous authors (12, 13) used serum-free media during treatment of cell cultures with aquaMEPHYTON. It will be of interest to determine if the presence of fetal bovine or other sera alters the effects of aquaMEPHYTON on cell lines and perhaps prevents the release of total protein from these cells. It is appropriate to mention that, although this report provides evidence for non-specific effects of aquaMEPHYTON on cells, this compound is apparently nontoxic to humans (22). We were able to find only one report of complications resulting from the administration of aquaMEPHYTON to humans (23). This case report described an association between intravenous administration of aquaMEPHYTON and acute development of hypotension and subsequent cardiovascular collapse (23). The authors suggested that vitamin Kl, or the agents used to emulsify vitamin Kl, may have been responsible for the vasodilation (23).
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ACKNOWLEDGEMENTS This work was supported by a Molecular and Cellular Biology Post-Doctoral Training Grant NIH HL07527 (C.S.J.), an NIH research grant HL38232 (S.J.F.D.) and The Pew Memorial Trust (S.J.F.D.). S.J.F.D. is a Pew Scholar in the Biomedical Sciences and an Established Investigator of the American Heart Association. REFERENCES
1.
Davie, E.W. and Fujikawa, K. Basic mechanisms Annu Rev Biochem 44,799-3291975.
2.
Davie, E.W., Fujikawa, K., Kurachi, K. and Kisiel, W. The role of serine proteases in the blood coagulation cascade. Adv Enzymol48,277-318, 1979.
3.
Jackson, C.M. and Nemerson, Y. Blood coagulation. 765811,198O.
4.
Kisiel, W. and Hanahan, D.J. Purification and characterization factor II. Biochim Biophys Acta 304, 103-113, 1973.
5.
Barnhart, M.I. 360-366,196O.
6.
Walz, D.A., Hewett-Emmett, D., Guillin, M.-C. Amino acid sequences and molecular homology of the vitamin K-dependent clotting factors. In: Prothrombin and Other Vitamin K Proteins, vol 1, W H Seegers and D A Walz (Eds.) Boca Raton, FL: CRC Press, Inc 1986, pp. 125-160.
7.
Stenflo, J., Fernlund, P., Egan, W. and Roepstorff, P. Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad Sci, USA 71,2730-2733,1974.
8.
Nelsestuen, G.L., Zytkovicz, T.H. and Howard, J.B. The mode of action of vitamin K: Identification of gamma-carboxyglutamic acid as a component of prothrombin. J Biol Chem 249,6347-6350,1974.
9.
Stenflo, J. and Suttie, J.W. Vitamin K-dependent formation of gammacarboxyglutamic acid. Annu Rev Biochem 46, 157-172,1977.
Annu Rev Biochem 49,
Cellular site for prothrombin synthesis.
10. Suttie, J.W. Vitamin K-dependent carboxylase. 477,1985. 11.
in blood coagulation.
of human
Am J Physiol 199,
Annu Rev Biochem 54, 459-
Esmon, C.T., Sadowski, J.A. and Suttie, J.W. A new carboxylation reaction: the vitamin K-dependent incorporation of H14CO3- into prothrombin. J Biol Chem 250,4744-4748,1975.
Vol. 65,No.3
419
VlTAMlNK,&PROTHROMBlNmRNALEVELS
12. Fair, D.S. and Bahnak, B.R.
Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III. Blood 64, 194-204, 1984.
13. Munns,
T.W., Johnston, M.F.M., Liszewski, M.K. and Olson, R.E.L. Vitamin K-dependent synthesis and modification of precursor prothrombin in cultured H-35 hepatoma cells. Proc iVutZ Acad Sci, USA 73, 2803-2807, 1976. Human hepatocellular B.B., Howe, C.C. and Aden, D.P. carcinoma cell lines secreted the major plasma proteins and hepatitis B surface antigen. Science 209,497-499, 1980.
14. Knowles,
15. Javitt, N.B. Hep G2 cells as a resource for metabolic studies: cholesterol, and bile acids. FASEB J4, 161-168, 1990.
lipoprotein,
16. Glisin, V., Crkvenjakov, R. and Byus, C. Ribonucleic acid isolated by cesium chloride centrimgation.
Biochemistry 13, 2633-2637, 1974.
17. Jamison,
C.S. and Degen, S.J.F. mRNA coding for rat prothrombin. 1991.
Prenatal and postnatal expression of Biochim Biophys Acta 1088, 208-216,
18. Degen, S.J.F., MacGillivray, R.T.F. and Davie, E.W. Characterization of the complementary deoxyribonucleic acid and prothrombin. Biochemistry 22, 2087-2097, 1983.
gene
coding
for
human
19. Feinberg,
A.P. and Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132,6-13,1983.
20. Feinberg,
A.P. and Vogelstein, B. ADDENDUM: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137,266-267, 1984.
21. Durnam, D.M. and Palmiter, R.D.
A practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem 131, 385-393, 1983.
22. Marcus, R. and Coulston, A.M.
Fat-soluble vitamins: Vitamins A, K, and E. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 8th edition, A G Gilman, T W Rall, A S Nies and P Taylor (Eds.) New York: Pergamon Press 1990, pp. 1563-1566.
23.
Barash, P., Kitahata, L.M. and Mandel, S. Acute cardiovascular collapse after intravenous phytonadione. Anesth Analg Curr Res 55, 304-306, 1976.
