Modulation of intracellular calcium and proliferative activity of invertebrate and vertebrate cells by ethylene
© Perovic et al; licensee BioMed Central Ltd. 2001
Received: 3 January 2001
Accepted: 31 May 2001
Published: 31 May 2001
Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase.
Here we describe for the first time, that besides sponge cells, mammalian cell lines (mouse NIH-3T3 and human HeLa and SaOS-2 cells) respond to ethylene, generated by ethephon, with an immediate and strong, transient increase in [Ca2+]i level, as demonstrated using Fura-2 imaging method. A rise of [Ca2+]i level was also found following exposure to ethylene gas of cells kept under pressure (SaOS-2 cells). The upregulation of [Ca2+]i was associated with an increase in the level of the cell cycle-associated Ki-67 antigen. In addition, we show that the effect of ethephon addition to S. domuncula cells depends on the presence of calcium in the extracellular milieu.
The results presented in this paper indicate that ethylene, previously known to act as a mediator (hormone) in plants only, deserves also attention as a potential signaling molecule in higher vertebrates. Further studies are necessary to clarify the specificity and physiological significance of the effects induced by ethylene in mammalian cells.
Ethylene is the chemically simplest plant hormone. This compound plays an important regulatory role in plant growth, development, and senescence; it is involved in a variety of stress responses in plants (for a review, see ). In the last years, much progress has been made in the isolation and characterisation of the genes and proteins participating in the ethylene signal transduction pathway in plants (for a review, see ). Calcium and protein phosphorylation/dephosphorylation processes may be involved in the transduction of the ethylene signal . Ethylene is also among the mediators of programmed cell death in plants .
Recently we demonstrated for the first time that besides plants, certain animal cells, namely cells from a marine sponge (Suberites domuncula), sensitively react to ethylene . This gas is present, at a concentration of up to 100 pM, in seawater , where it can be produced from dissolved organic carbon by photochemical (especially ultraviolet light-induced) reactions [7,8,9]. We showed that primmorphs of S. domuncula, consisting of aggregates of dissociated sponge cells that are able to proliferate , respond to ethylene with an increase in [Ca2+]i and a reduction of apoptosis induced by starvation . In addition, in S. domuncula primmorphs an upregulation of the expression of two genes occurs following ethylene exposure, one of these genes, termed SDERR , is related to the ethylene-responsive plant gene HEVER . The other gene encodes the Ca2+/calmodulin-dependent protein kinase II . The SDERR cDNA has been isolated and characterized .
The sponges (Porifera) are considered to form the first or one of the first metazoan phyla that diverged from the common ancestor of all Metazoa, the Urmetazoa . They are provided with the same protein constituents known from higher animals, including molecules involved in cell recognition and signal transduction pathways (for a review, see ). Therefore, we asked if besides sponges, cells from higher vertebrates respond to ethylene. Here we demonstrate that various mammalian cell lines react to ethylene, generated by ethephon (or ethylene gas), with an upregulation of [Ca2+]i level and an increased expression of the cell cycle-associated antigen Ki-67, used as a marker of cell proliferation.
Effect of ethylene on [Ca2+]i level in mammalian cells
Decomposition of ethephon in solution at increasing pH results in the production of H3PO4. Therefore, in control experiments, 0.5 mM of H3PO4 was added instead of ethephon (Figure 2A). The results revealed that H3PO4 did not cause any change of [Ca2+]i level. Similarly no change in [Ca2+]i level was found in assays without ethephon or any other compounds (not shown).
The extent of upregulation of [Ca2+]i level following ethylene exposure considerably varied between the cell lines studied. Only a relatively small but significant (p < 0.001) increase in [Ca2+]i level (increase in 340/380 nm ratio from 0.66 ± 0.01 to 0.74 ± 0.01) was observed in HeLa cells treated with a high concentration of 2 mM ethephon (Figure 2B), while osteosarcoma SaOS-2 cells responded to ethylene exposure (increase in 340/380 nm ratio from 0.90 ± 0.01 to 1.03 ± 0.05) already at a low concentration of 0.3 mM ethephon (Figure 2C). Exposure of cells (SaOS-2 cells) to ethylene (generated by ethephon addition) in Locke's solution depleted of CaCl2 resulted in only a small but not significant effect on [Ca2+]i level, indicating that the response of mammalian cells requires the presence of extracellular calcium (results not shown).
Effect of ethylene on mammalian cell proliferation
Effect of ethylene on proliferative activity of mammalian cells. The proliferative activity of the cells is expressed as Ki-67 labeling index. Results are means ± S.D. of 8 independent experiments.
Ki-67 labeling index (%)
Addition of ethephon (1 mM)
42 ± 14
68 ± 11**
38 ± 6
47 ± 6*
37 ± 7
49 ± 10*
Effect of ethylene on [Ca2+]i level in sponge cells
Effect of ethephon on cell viability
In the concentration range used ethephon displayed no effect on cell viability as determined in L5178y mouse lymphoma cells and HeLa cells. The cells were grown in the presence of 0.001 to 1000 μM ethephon. The ED50 was >1 mM.
Previously we found that low concentrations of ethylene present in seawater  significantly reduce the extent of apoptosis caused by starvation in primmorphs of the marine sponge S. domuncula . Primmorphs, which contain proliferating cells, are formed by aggregation of dissociated sponge cells . In invertebrate (sponge) cells, the mode of action of ethylene, a well-known growth hormone in plants (for a review, see ), is still uncertain but seems to be associated with calcium metabolism .
In plants, ethylene has important regulatory functions; its production can be elicited by various stress factors (for a review, see ), including stress by oxygen radicals [15, 16]. The molecular basis of the ethylene signal transducing system has been studied mainly in Arabidopsis (for reviews, see [17,18,19]). A specific receptor to which ethylene binds has been identified .
In view of our finding that ethylene responsive pathways exist in sponges, the phylogenetic oldest metazoan phylum, we examined if cells derived from higher Metazoa (human and animal cell lines) respond to ethylene too. This alkene was produced in the culture fluid by addition of ethephon , which hydrolyzes in aqueous solution at pH >3.5 under formation of ethylene .
Using the Fura-2 imaging method, we could demonstrate that all mammalian cell lines studied reacted to ethylene, generated from ethephon, by an increase in [Ca2+]i level. The rise of [Ca2+]i level occurred immediately after addition of ethephon to the medium, indicating that ethylene causes a fast effect on cell metabolism. However, the threshold value and the extent of the ethylene-induced effect on [Ca2+]i level strongly differed among the cells examined. NIH-3T3 fibroblasts and osteosarcoma SaOS-2 cells sensitively responded to lower concentrations of ethylene produced by 0.3 mM ethephon, while the effect on HeLa cells was rather weak but significant (at 1 and 2 mM ethephon). In addition the amount of cells responding to ethylene within the total cell population varied among the cell lines examined.
Exposure of mammalian cells kept under pressure (SaOS-2 cells) to ethylene gas, instead of ethylene generated by ethephon, caused also a strong but more protracted increase in [Ca2+]i level compared to ethephon addition, most likely due to the different kinetics of changes of concentration of dissolved ethylene. This result and the finding that H3PO4 generated during ethephon hydrolysis does not change [Ca2+]i level demonstrate that the effects observed are specifically induced by ethylene. In addition, ethephon displayed no effect on cell viability.
The increase in [Ca2+]i level induced by ethylene exposure may be associated with changes in cell proliferation. Therefore, as a measure of the proliferative activity of the cells, the Ki-67 labeling index was determined. The Ki-67 antigen, a dimeric, non-histone protein with a molecular weight of 345-395 kDa is specifically expressed by proliferating cells; it is absent in resting cells . Using the Ki-S5 antibody, we determined that the expression of Ki-67 was significanly increased following ethylene exposure, indicating that ethylene may activate cell proliferation. At this time point (10 h after treatment with ethylene) [Ca2+]i has recovered basal levels (not shown in Figure 2A,B,C).
The concentrations at which the ethylene-releasing agent ethephon induced a shift of [Ca2+]i level in mammalian cells are in the same range or close to that at which sponge cells reacted (1 mM ethephon). However, the increase in [Ca2+]i level induced by this compound in at least some mammalian cell lines was even stronger than in sponge cells. From these results we conclude that besides invertebrate (sponge) cells, mammalian cells are sensitive to ethylene. In addition, we could demonstrate that the response of sponge cells following ethylene exposure depends on the presence of Ca2+ in the surrounding medium; no effect was observed if this metal ion was absent in the external milieu.
At present it is unknown if ethylene binds to a membrane receptor in mammalian cells, and sponge cells too. Therefore, the mechanism of the effect of ethylene resulting in an increase in intracellular level of calcium, one important messenger in intracellular signal transduction pathways, is still unclear. In sponge cells a Ca2+/calmodulin-dependent protein kinase II is up-regulated after ethylene exposure . In mammalian cells, it is known that Ca2+ mediates the Ca2+/calmodulin-dependent protein kinase II cascade , resulting in prevention of apoptosis . The second gene which was found to be upregulated in sponges after ethylene exposure is the proposed ethylene-responsive gene, SDERR, which has been isolated from S. domuncula .
It should be noted that the minimal concentration of ethylene required to evoke the raise of [Ca2+]i level in mammalian cells (3.2 μM) is rather high compared to the threshold concentration in plants, which may be as low as 0.1 μl/liter; this corresponds to an aqueous solution of 0.65 nM ethylene at 25°C . Therefore it cannot be excluded that the ethylene-induced effects in mammalian cells are non-specific due to the anesthetic effect of the gas which is not mediated by a receptor. Future studies have to show whether animal cells possess a specific receptor for ethylene gas as found in plant cells . These studies are necessary to demonstrate the physiological significance of the observed effects.
In summary, our results show that mammalian cells respond to ethylene with an increase in [Ca2+]i and cell proliferation (increased expression of cell-cycle-associated Ki-67 protein). In sponges the effects of ethylene on cell physiology are associated with an upregulation of an ethylene-responsive gene, SDERR . At present, it is unknown if similar proteins exist also in cells from higher animals and humans.
Measurements of intracellular calcium level ([Ca2+]i) in various mammalian cell lines (mouse NIH-3T3 and human HeLa and SaOS-2 cells) revealed that ethylene, produced by ethephon, caused a significant upregulation of [Ca2+]i in these cells. A similar effect was found in cells kept under pressure after exposure to ethylene gas. These data support previous findings showing an upregulation of [Ca2+]i, as well as an increased expression of an ethylene-responsive gene, SDERR, in invertebrate cells (primmorphs of the marine sponge S. domuncula) . These results indicate that ethylene is not only an important mediator of many biological processes in plants but may also have some modulatory effects on intracellular signaling pathways in animals.
Materials and Methods
Fura-2-acetoxymethyl ester (Fura-2-AM) was obtained from Molecular Probes (Leiden, The Netherlands); (2-chloroethyl)phosphonic acid (ethephon), (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue) (MTT) and natural Ca2+- and Mg2+-containing seawater (SW) were purchased from Sigma (Deisenhofen, Germany); and anti-Ki-67 (Ki-S5) mouse monoclonal antibody was from Roche Diagnostics (Mannheim, Germany). The sources of all other chemicals used were as described previously [26, 27]. The composition of Ca2+- and Mg2+-free artificial seawater (CMFSW) was given earlier .
Mammalian cells and sponge primmorphs
Mouse embryonic NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium containing streptomycin, penicillin and 10% fetal calf serum (FCS). HeLa cells were propagated in RPMI 1640 medium with 10% FCS. Human osteosarcoma SaOS-2 cells were cultivated in McCoy's 5A medium, supplemented with 1.5 mM L-glutamine, 0.1 g/l gentamicin and 15% FCS. All cell cultures were kept in a fully humidified atmosphere and 5% CO2/air at 37°C.
Specimens of the marine sponge Suberites domuncula (Porifera, Demospongiae, Hadromerida) were collected in the Northern Adriatic near Rovinj (Croatia) and kept in aquaria in Mainz (Germany) at a temperature of 17°C. Sponge primmorphs were prepared from dissociated sponge cells as described .
Ethephon was dissolved in 1x phosphate-buffered saline pH 2.5 yielding a stock solution of 69 mM and kept at 4°C. At this pH, an aqueous solution of ethephon is stable. At higher pH values (> pH 3.5), ethephon hydrolyses under formation of free ethylene and phosphoric acid (H3PO4) .
The concentration of intracellular calcium ([Ca2+]i) was determined using the Ca2+-indicator dye Fura-2-AM; the fluorescence ratio at 340 and 380 nm was measured as described [29, 30]. "Chambered coverglass" incubation chambers (Lab-Tek, Nunc) were coated with poly-L-lysine. Mammalian cells were loaded in the dark with 6-8 μM Fura-2-AM in medium containing 1% FCS and 1% bovine serum albumin at 37°C for 1 h; sponge cells (primmorphs) were loaded with 10-12 μM Fura-2-AM in CMFSW containing 1% bovine serum albumin at 17°C for 2 h. Subsequently, the cells were washed twice with medium supplemented with 10% (HeLa and NIH-3T3 cells) or 15% FCS (SaOS-2 cells) and incubated further at 37°C for 1 h; sponge cells were washed with CMFSW and incubated further at 17°C for 1 h. These time periods were sufficient to load the cells with Fura-2-AM (inactive Fura-2) and for hydrolysis of the acetoxymethyl ester (active Fura-2). For the experiments mammalian cells were incubated with ethephon (0.1 to 2 mM) in Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 5.6 mM glucose and 10 mM Hepes, pH 7.4); sponge cells were incubated in SW or CMFSW. Treatment of the cells with ethephon was performed 5 min after starting measurement of [Ca2+]i, which proceeded for 13.5 min. Control assays were performed by addition of 0.5 mM H3PO4 instead of ethephon. Fluorescence images were analysed using an inverted-stage Olympus IX70 microscope and a computerized imaging system as described before . Calibration was performed with the "Fura-2 Calcium Imaging Calibration Kit" according to the instructions of the manufacturer (Molecular Probes). One unit of fluorescence ratio 340/380 nm equals ≈ 143 nM [Ca2+]i.
To determine the effect of ethylene gas on [Ca2+]i of SaOS-2 cells, cells on coverglass (2 × 105 cells/cm2 in 100 μl medium; after loading with Fura-2-AM and activation as above; diameter of the glass plates, 1 cm) were transferred into a pressure chamber as described previously . The pressure in the chamber was generated by air (1 atm). Varying amounts of ethylene gas in air were injected using a syringe (10 ml; approximately a 5-fold excess compared to total gas space of the system) 5 min after starting measurement of [Ca2+]i, which was performed for 20 min.
The amount of ethylene dissolved in medium within the pressure chamber after injection of ethylene gas was determined in a Shimadzu GC9A apparatus using a FID detector. A column (size 3 × 600 mm) filled with aluminium oxide was used and the runs were performed at 70°C.
Immunostaining of Ki-67 antigen
Cells are fixed with 4% paraformaldehyde and incubated with anti-Ki-67 (Ki-S5) antibody for 30 min at room temperature. After washing, the cells were incubated with anti-mouse Ig-fluorescein for 30 min at room temperature. Analysis was performed by fluorescence microscopy. The proliferative activity of the cells is expressed as the percentage of Ki-67 positive cells over the total number of cells.
The viability of HeLa cells and L5178y mouse lymphoma cells  was determined by the MTT colorimetric assay . Evaluation (at 595 nm) was performed using an ELISA plate reader (BioRad 3550) equipped with the program NCIMR IIIB. The effective dose, which inhibits cell proliferation by 50% (ED50) was estimated by logit regression .
The results were analyzed by Student's t-test .
intracellular concentration of Ca2+
Ca2+ and Mg2+-free artificial seawater
fetal calf serum
3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
Ca2+- and Mg2+-containing seawater.
We would like to thank Dr. Peter Beutelmann, Institut für Allgemeine Botanik, Universität Mainz, for his help with gas chromatography. This work was supported by grants from the Boehringer Ingelheim Foundation and the International Human Frontier Science Program (RG-333/96-M; W.E.G. M.).
- Ecker JR: The ethylene signal transduction pathway in plants. Science. 1995, 268: 667-675.View ArticlePubMedGoogle Scholar
- Solano R, Ecker JR: Ethylene gas: perception, signaling and response. Curr Opin Plant Biol. 1998, 1: 393-398. 10.1016/S1369-5266(98)80262-8.View ArticlePubMedGoogle Scholar
- Kwak SH, Lee SH: The requirements for Ca 2+, protein phosphorylation, and dephosphorylation for ethylene signal transduction in Pisum sativum L. Plant Cell Physiol. 1997, 38: 1142-1149.View ArticlePubMedGoogle Scholar
- Lam E, Pontier D, del Pozo O: Die and let live - programmed cell death in plants. Curr Opin Plant Biol. 1999, 2: 502-507. 10.1016/S1369-5266(99)00026-6.View ArticlePubMedGoogle Scholar
- Krasko A, Schröder HC, Perovic S, Steffen R, Kruse M, Reichert W, Müller IM, Müller WEG: Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis. J Biol Chem. 1999, 274: 31524-31530. 10.1074/jbc.274.44.31524.View ArticlePubMedGoogle Scholar
- Ratte M, Bujok O, Spitzy A, Rudolph J: Photochemical alkene formation in seawater from dissolved organic carbon: results from laboratory experiments. J Geophys Res. 1998, 103: 5707-5717.View ArticleGoogle Scholar
- Wilson DJ, Swinnerton JW, Lamontagne RA: Production of carbon monoxide and gaseous hydrocarbons in seawater: relation to dissolved organic carbon. Science. 1970, 168: 1577-1579.View ArticlePubMedGoogle Scholar
- Ferek R, Andreae MO: Photochemical production of carbonyl sulfide in marine surface waters. Nature. 1984, 307: 148-150.View ArticleGoogle Scholar
- Zuo Y, Jones RD: Formation of carbon monoxide by photolysis of dissolved marine organic material and its significance in the carbon cycling of the oceans. Naturwissenschaften. 1995, 82: 472-474. 10.1007/s001140050217.View ArticleGoogle Scholar
- Müller WEG, Wiens M, Batel R, Steffen R, Borojevic R, Custodio MR: Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula. Marine Ecol Progr Ser. 1999, 178: 205-219.View ArticleGoogle Scholar
- Sivasubramaniam S, Vanniasingham VM, Tan CT, Chua NH: Characterization of HEVER, a novel stress-induced gene from Hevea brasiliensis. Plant Mol Biol. 1995, 29: 173-178.View ArticlePubMedGoogle Scholar
- Müller WEG, Müller IM: How was the Protozoa - Metazoa thresholdcrossed: The Urmetazoa. Comp Biochem Physiol. 2000, 126/B (Suppl 1): S69-View ArticleGoogle Scholar
- Müller WEG: Molecular phylogeny of Metazoa (animals): monophyletic origin. Naturwissenschaften. 1995, 82: 321-329. 10.1007/s001140050190.View ArticlePubMedGoogle Scholar
- Abeles FB, Morgan PW, Saltveit ME: Ethylene in Plant Biology. San Diego: Academic Press,. 1992Google Scholar
- Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR: EINe, a bifunctional transducer ethylene and stress response in Arabidopsis. Science. 1999, 284: 2148-2152. 10.1126/science.284.5423.2148.View ArticlePubMedGoogle Scholar
- Woeste K, Kieber JJ: A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response as well as a rosette-lethal phenotype. Plant Cell. 2000, 12: 443-455. 10.1105/tpc.12.3.443.PubMed CentralView ArticlePubMedGoogle Scholar
- Kieber JJ: The ethylene response pathway in Arabidopsis. Annu Rev Plant Physiol Plant Mol Biol. 1997, 48: 277-296. 10.1146/annurev.arplant.48.1.277.View ArticlePubMedGoogle Scholar
- Johnson PR, Ecker JR: The ethylene gas signal transduction pathway: a molecular perspective. Annu Rev Genet. 1998, 32: 227-254. 10.1146/annurev.genet.32.1.227.View ArticlePubMedGoogle Scholar
- Savaldi-Goldstein S, Fluhr R: Signal transduction of ethylene perception. In Results and Problems in Cell Differentiation. Edited by Hirt H. Berlin: Springer-Verlag,. 2000, : 145-161.Google Scholar
- Bleecker AB, Estelle MA, Somerville C, Kende H: Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science. 1988, 241: 1086-1089.View ArticlePubMedGoogle Scholar
- Cooke AR, Randall DI: 2-Haloethanephosphonic acids as ethylene releasing agents for the induction of flowering in pineapples. Nature. 1968, 218: 974-975.View ArticlePubMedGoogle Scholar
- Yang SF: Ethylene evolution from 2-chloroethylphosphonic acid. Plant Physiol. 1969, 44: 1203-1204.PubMed CentralView ArticlePubMedGoogle Scholar
- Gerdes J, Li L, Schlueter C, Duchrow M, Wohlenberg C, Gerlach C, Stahmer I, Kloth S, Brandt E, Flad HD: Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 1991, 138: 867-873.PubMed CentralPubMedGoogle Scholar
- Hardie G, Hanks S: The Protein Kinase FactBooks: Protein-Serine Kinases. London: Academic Press,. 1995Google Scholar
- Yano S, Tokumitsu H, Soderling TR: Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature. 1998, 396: 584-587. 10.1038/25147.View ArticlePubMedGoogle Scholar
- Seack J, Kruse M, Müller IM, Müller WEG: Promoter and exon-intron structure of the protein kinase C gene from the marine sponge Geodia cydonium : evolutionary considerations and promoter activity. Biochim Biophys Acta. 1999, 1444: 241-253. 10.1016/S0167-4781(98)00275-9.View ArticlePubMedGoogle Scholar
- Wimmer W, Perovic S, Kruse M, Krasko A, Batel R, Müller WEG: Origin of the integrin-mediated signal transduction: functional studies with cell cultures from the sponge Suberites domuncula. Eur J Biochem. 1999, 260: 156-165. 10.1046/j.1432-1327.1999.00146.x.View ArticlePubMedGoogle Scholar
- Rottmann M, Schröder HC, Gramzow M, Renneisen K, Kurelec B, Dorn A, Friese U, Müller WEG: Specific phosphorylation of proteins in the pore complex-lamina from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. EMBO J. 1987, 6: 3939-3944.PubMed CentralPubMedGoogle Scholar
- Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca 2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985, 260: 3440-3450.PubMedGoogle Scholar
- Perovic S, Wichels A, Schütt C, Gerdts G, Pahler S, Steffen R, Müller WEG: Neuroactive compounds produced by bacteria from the marine sponge Halichondria panicea : activation of the neuronal NMDA receptor. Environm Toxicol Pharmacol. 1998, 6: 125-133. 10.1016/S1382-6689(98)00028-3.View ArticleGoogle Scholar
- Müller WEG, Geisert M, Zahn RK, Maidhof A, Bachmann M, Umezawa H: Potentiation of the cytostatic effect of bleomycin on L5178y mouse lymphoma cells by pepleomycin. Eur J Cancer Clin Oncol. 1983, 19: 665-670.View ArticlePubMedGoogle Scholar
- Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR: Evaluation of a tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988, 48: 4827-4833.PubMedGoogle Scholar
- Sachs L: Angewandte Statistik. Berlin: Springer,. 1997Google Scholar
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