J Cancer Res Clin Oncol (1991) 117:37-42
Caiicer ~esearch Clinical 9
017152169100008U
@ Springer-Verlag 1991
Influence of gastrin, gastrin receptor blockers, epidermal growth factor, and difluoromethylornithine on the growth and the activity of ornithine decarboxylase of colonic carcinoma cells * S. Eggstein 1, A. ImdahP, M. Kohler 2, M. Waibel 1, and E.H. Farthmann 1 1 Chirurgische Universitfitsklinik Freiburg, Abteilung Allgemeine Chirurgie und Poliklinik, Hugstetterstrasse 55, W-7800 Freiburg i. Br., Federal Republic of Germany 2 Institut fiir Biologie 2, Schfinzlestrasse, W-7800 Freiburg, Federal Republic of Germany Received 29 June 1990/Accepted 6 September 1990
Summary. Polyamines are essential factors of cell growth
Key words: Ornithine decarboxylase - 2-difluoromethyl-
and differentiation. Modulation of the cellular polyamine content by 2-difluoromethylornithine (DFMO) inhibiting ornithine decarboxylase (ODC), or by hormones inducing ODC, influences cell growth. Gastrin acts trophically on some colonic carcinomas and their growth is inhibited by gastrin receptor blockers. The mechanism of the trophic action of gastrin on colonic carcinomas is not known. In this study the effect of gastrin, gastrin receptor blockers, epidermal growth factor (EGF) and DFMO on growth and ODC activity of four human colon carcinoma cell lines (SW 403, SW 1116, LS 174 T and Lovo) was investigated. Growth and ODC activity of all cell lines were inhibited by DFMO. Growth of the SW 403 cell line was increased by gastrin and inhibited by the gastrin receptor blocker benzotrypte. The other cell lines did not respond to gastrin and the gastrin receptor blocker. In SW 403 cells ODC activity was increased by gastrin, and was also elevated after treatment with the gastrin receptor blocker. These in vitro results were confirmed by studies on tumours that developed from SW 403 cells in nude mice. Combination of benzotrypte and DFMO did not enhance the antiproliferative effect. EGF increased growth of SW 403 cells, but no induction of ODC activity was measured. LS 174 T cells were not stimulated by EGF. Medium replacement was the strongest stimulus of ODC activity in SW 403 cells already inducing ODC after 3 h. During cell culture ODC activity was high after seeding and decreased continuously with increasing cell density. These data suggest that gastrin induces ODC in gastrin-sensitive colonic carcinoma cells. DFMO appears to be a valuable antiproliferative agent in colonic carcinoma cells.
ornithine - Colonic carcinoma cell lines trin receptor blockers EGF
* Supported by Deutsche Forschungsgemeinschaft(DFG) Abbreviations: ODC, Ornithine decarboxylase; DFMO, 2-di-
fluoromethylornithine;EGF, epidermalgrowth factor Offprint requests to: S. Eggstein
Gastrin - Gas-
Introduction The polyamines putrescine, spermidine and spermine are essential factors of cell proliferation and differentiation. In many neoplasms polyamine concentrations are elevated, and the concentration ratio of polyamines differs from that of normal tissue (Kingsnorth et al. 1984; Lundell and Rosengren 1986; Malt et al. 1985). Therefore, a connection has been suggested between neoplastic proliferation and intracellular polyamine levels. Polyamine depletion decreases cell proliferation, and is able to impair the expression of oncogens (Celano et al. 1988). The biosynthesis ofpolymines from ornithine is controlled by ornithine decarboxylase (ODC), the key enzyme of polymine synthesis generating putrescine. Spermidine and spermine arise from putrescine (Pegg and McCann 1982). Hormones (Parko and Kenny 1971; Maudsley et al. 1976; Rosewicz et al. 1988) and growth factors (Fitzpatrick et al. 1987) induce ODC and have atrophic effect on their target organs by increasing the concentration of polyamines. On the other hand, the decrease of the cellular concentrations of polyamines caused by 2-difluoromethylornithine (DFMO), an irreversible "suicide inhibitor" of ODC, significantly inhibits the cell proliferation (Malt et al. 1985). This proliferation-inhibiting effect of DFMO has been shown in carcinogen-induced colonic carcinomas of the rat (Zhang et al. 1988) and xenotransplantated human colon carcinomas in nude mice (Tutton et al. 1986). Gastrin has atrophic effect on the oxyntic gland and colon mucosa (Johnson 1981), as well as on certain carcinomas of the colon. Gastrin-receptor-blocking agents inhibit the growth of these gastrin-sensitive carcinomas (Imdahl et al. 1989; Eggstein and Imdahl 1988). Because the trophic effect of gastrin is at least partially inhibited
38 b y D F M O (Seidel et al. 1985), a c o n n e c t i o n with p o l y a m i n e m e t a b o l i s m is suggested. A t least in the colon, no i n d u c t i o n o f O D C b y gastrin has been i n d i c a t e d (Seidel et al. 1985; F i t z p a t r i c k et al. 1987). Recently, J o h n s o n et al. (1988) f o u n d i n c r e a s e d c o n c e n t r a t i o n s o f O D C in a s u b p o p u l a t i o n o f r a t o x y n t i c g l a n d cells after t r o p h i c s t i m u l a t i o n with gastrin, using a n t i s e r u m - d i l u t i o n techniques. I n v e s t i g a t i o n s c o n c e r n i n g the influence o f g a s t r i n o n O D C activity in c o l o n i c c a r c i n o m a o r h u m a n c o l o n m u c o s a h a v e n o t yet been p u b l i s h e d . P r o v e d k n o w l e d g e o f a p o s s i b l e influence o f gastrin o r g a s t r i n - r e c e p t o r - b l o c k ing agents o n O D C activity in c o l o n c a n c e r c o u l d shed new light o n t h e r a p y . I n this r e p o r t we p r e s e n t results o n the influence o f gastrin, e p i d e r m a l g r o w t h f a c t o r ( E G F ) , the gastrin rec e p t o r b l o c k e r b e n z o t r y p t e a n d D F M O o n the O D C activity a n d the g r o w t h o f f o u r h u m a n c o l o n i c c a r c i n o m a cell lines in vitro a n d p a r t l y in x e n o t r a n s p l a n t s o n n u d e mice. O u r results s h o w t h a t O D C activity is s t i m u l a t e d b y g a s t r i n a n d gastrin r e c e p t o r b l o c k e r s in the gastrin-sensitive c o l o n c a r c i n o m a cell line S W 403 in vitro a n d in the n u d e mice m o d e l . O D C activity a n d g r o w t h o f all cell lines were i n h i b i t e d b y D F M O .
Materials and methods Cell lines. The human colonic-carcinoma-derived cell lines SW 403, LS 174 T, Lovo and SW 1116 of the American Type Culture Collection (kindly supplied by the lnstitnt ffir Immunbiologie, Freiburg, FRG) were cultured in Dulbecco's or RPM I medium with 10 % fetal calf serum and antibiotics in 10-cm petri dishes containing 10 ml medium at 37~ C, 95% air and 5% CO2. Medium was replaced every 2nd or 3rd day. Cells were in each case subcultivated before growing confluently. After trypsination (0.03% trypsin in Hank's EDTA, 30 min, 37 ~ C) and eosin staining for viability 7 x 104 3 x 105 cells/ml were seeded. Cells were counted in a Neubauer cytometer. Cell culture materials were purchased from Gibco, USA. The following substances were added to the media (without preincubation): 10 ~tg/ml pentagastrin (Gastrodiagnost, Merck, Darmstadt, FRG), 8 Ixg/ml benzotrypte (kindly donated by Dr. Rovati, Rotta Research, Milano, italy), 8 ~tg/ml DFMO (kindly donated by Merell Dow Research, StraBburg, France), benzotrypte with DFMO in the concentrations mentioned above, 5 ng/ml EGF (Sigma, St. Louis, USA). Cell number and ODC were determined after 3 days and 8 days. In order to investigate the influence of medium replacement on ODC activity, SW 403 cells were preincubated over 48 h, the medium was replaced, and ODC was determined after the periods described below. Nude mice experiments. Experiments were carried out as already described (Imdahl et al. 1989): 105, SW 403 and LS 174 T cells were injected subcutaneously. The developing tumours were pooled and assayed for ODC activity after 4 weeks of treatment with 60 gg pentagastrin and 100 ~tg proglumide (kindly donated by Dr. Rovati, Rotta Research, Milano, Italy) and 0.9% NaC1 as control injected twice daily intraperitoneally. Determination of ODC activity. The assay was modified according to Bauknecht et al. (1987). All procedures were carried out in ice. Tumours were immediately frozen in liquid nitrogen and stored at - 7 0 ~ C. Cells were washed in 0.9% NaC1 after trypsination and the 200g (4 ~ C, 10 min) pellet was frozen. After homogenisation with the Ultratur-
rax homogeniser (IKA, Staufen, FRG) in 1.5 ml/g extraction buffer [0.1 M HEPES, 9 mM dithioerithritol (Aldrich, Steinheim, FRG), 5.0 mM EDTA (Merck, Darmstadt, FRG), 1.2 mM pyridoxal 5'phosphate (Sigma, St. Louis, USA), pH 6,8] three samples of the 35000 g supernatant (4~ C, 20 rain) were assayed for ODC and protein, by the method of Lowry et al. (1951). Assay tubes containing 400 gl supernatant, 390 gl extraction buffer and 10 nmol [xgCOz]ornithine (Amersham, Buckinghamshire, England) in 10 ~tl aqueous solution with 2% ethanol were placed in scintillation vials with 2 ml phenylethylamine (Aldrich, Steinheim, FRG), capped, and incubated 60 rain at 37~ C. The reaction was terminated and the liberated 14CO2 expelled by adding 250 ~tl 20% trichloroacetic acid by means of an injection cannula through the closed cap. After another 2 h incubation the reaction tubes were removed, and the scintillation vials were filled with 10 ml scintillation fluid (Rotiszint, Roth, Karlsruhe, FRG). The liberated 14CO2 was estimated in a liquid scintillation counter (Rackbeta, LKB, Bromma, Sweden). The activity of ODC was assayed as liberated 1~CO2 in pmol mg protein-1 h - t as a mean value of three samples. For statistics the Student t-test was used.
Results S W 403 cells
T h e g r o w t h o f S W 403 cells was significantly ( P < 0 . 0 1 ) s t i m u l a t e d b y p e n t a g a s t r i n (Table 1). O n the 3rd d a y the g a s t r i n - s t i m u l a t e d cells h a d m u l t i p l i e d five times, w h e r e a s the c o n t r o l cells h a d m u l t i p l i e d f o u r times only. 'The O D C activity i n c r e a s e d f r o m 25 p m o l h - 1 m g - 1 in c o n t r o l to 50 p m o l h -1 m g -1 w h e n p e n t a g a s t r i n was added. T h e g a s t r i n r e c e p t o r b l o c k e r b e n z o t r y p t e significantly i n h i b i t e d the g r o w t h o f S W 403 cells ( P < 0 . 0 1 ) ; the cell p r o l i f e r a t i o n was r e d u c e d to 80% o f c o n t r o l after 3 days. C o m p a r e d w i t h the c o n t r o l , a p p l i c a t i o n o f b e n z o t r y p t e resulted in the d o u b l i n g o f O D C activity after 3 days. D F M O r e d u c e d the O D C activity to 6 0 % o f control. Cell g r o w t h was r e d u c e d to the s a m e degree after 3 days. C o m b i n a t i o n o f D F M O a n d b e n z o t r y p t e h a d the s a m e effect as the a d d i t i o n o f one s u b s t a n c e alone. A p p l i c a t i o n o f E G F resulted in a m a r k e d increase o f cell g r o w t h (Table 2). W e o b s e r v e d a 3 0 % increase o f
Table 1. Influence of pentagastrin, benzotrypte, difluoromethylornithine (DFMO) and benzotrypte together with DFMO on ornithine decarboxylase (ODC) activity and growth of SW 403 cells" Treatment
10 5 x Cell growth by day 3 (cells/ml)
ODC activity on day 3 (pmol h - 1 mg protein- 1)
Control Pentagastrin (10 gg/ml) Benzotrypte (8 gg/ml) DFMO (8 ~tg/ml) Benzotrypte + DFMO
5.5 __0.2 6.7 • 0.2 *
25 • 2.3 50 + 0.4 *
4.4 • 0.4 *
42 _+5.4 *
3.6 + 0.2 *
15 + 0.8 *
3.8•
i1 -I-0.3 *
*
" Values represent means + standard deviation of four experiments. Each group contained six culture plates. Initial concentration of cells on day 0 = 1.33 x 105 cells/ml * Significant difference (P
Caiicer ~esearch Clinical 9
017152169100008U
@ Springer-Verlag 1991
Influence of gastrin, gastrin receptor blockers, epidermal growth factor, and difluoromethylornithine on the growth and the activity of ornithine decarboxylase of colonic carcinoma cells * S. Eggstein 1, A. ImdahP, M. Kohler 2, M. Waibel 1, and E.H. Farthmann 1 1 Chirurgische Universitfitsklinik Freiburg, Abteilung Allgemeine Chirurgie und Poliklinik, Hugstetterstrasse 55, W-7800 Freiburg i. Br., Federal Republic of Germany 2 Institut fiir Biologie 2, Schfinzlestrasse, W-7800 Freiburg, Federal Republic of Germany Received 29 June 1990/Accepted 6 September 1990
Summary. Polyamines are essential factors of cell growth
Key words: Ornithine decarboxylase - 2-difluoromethyl-
and differentiation. Modulation of the cellular polyamine content by 2-difluoromethylornithine (DFMO) inhibiting ornithine decarboxylase (ODC), or by hormones inducing ODC, influences cell growth. Gastrin acts trophically on some colonic carcinomas and their growth is inhibited by gastrin receptor blockers. The mechanism of the trophic action of gastrin on colonic carcinomas is not known. In this study the effect of gastrin, gastrin receptor blockers, epidermal growth factor (EGF) and DFMO on growth and ODC activity of four human colon carcinoma cell lines (SW 403, SW 1116, LS 174 T and Lovo) was investigated. Growth and ODC activity of all cell lines were inhibited by DFMO. Growth of the SW 403 cell line was increased by gastrin and inhibited by the gastrin receptor blocker benzotrypte. The other cell lines did not respond to gastrin and the gastrin receptor blocker. In SW 403 cells ODC activity was increased by gastrin, and was also elevated after treatment with the gastrin receptor blocker. These in vitro results were confirmed by studies on tumours that developed from SW 403 cells in nude mice. Combination of benzotrypte and DFMO did not enhance the antiproliferative effect. EGF increased growth of SW 403 cells, but no induction of ODC activity was measured. LS 174 T cells were not stimulated by EGF. Medium replacement was the strongest stimulus of ODC activity in SW 403 cells already inducing ODC after 3 h. During cell culture ODC activity was high after seeding and decreased continuously with increasing cell density. These data suggest that gastrin induces ODC in gastrin-sensitive colonic carcinoma cells. DFMO appears to be a valuable antiproliferative agent in colonic carcinoma cells.
ornithine - Colonic carcinoma cell lines trin receptor blockers EGF
* Supported by Deutsche Forschungsgemeinschaft(DFG) Abbreviations: ODC, Ornithine decarboxylase; DFMO, 2-di-
fluoromethylornithine;EGF, epidermalgrowth factor Offprint requests to: S. Eggstein
Gastrin - Gas-
Introduction The polyamines putrescine, spermidine and spermine are essential factors of cell proliferation and differentiation. In many neoplasms polyamine concentrations are elevated, and the concentration ratio of polyamines differs from that of normal tissue (Kingsnorth et al. 1984; Lundell and Rosengren 1986; Malt et al. 1985). Therefore, a connection has been suggested between neoplastic proliferation and intracellular polyamine levels. Polyamine depletion decreases cell proliferation, and is able to impair the expression of oncogens (Celano et al. 1988). The biosynthesis ofpolymines from ornithine is controlled by ornithine decarboxylase (ODC), the key enzyme of polymine synthesis generating putrescine. Spermidine and spermine arise from putrescine (Pegg and McCann 1982). Hormones (Parko and Kenny 1971; Maudsley et al. 1976; Rosewicz et al. 1988) and growth factors (Fitzpatrick et al. 1987) induce ODC and have atrophic effect on their target organs by increasing the concentration of polyamines. On the other hand, the decrease of the cellular concentrations of polyamines caused by 2-difluoromethylornithine (DFMO), an irreversible "suicide inhibitor" of ODC, significantly inhibits the cell proliferation (Malt et al. 1985). This proliferation-inhibiting effect of DFMO has been shown in carcinogen-induced colonic carcinomas of the rat (Zhang et al. 1988) and xenotransplantated human colon carcinomas in nude mice (Tutton et al. 1986). Gastrin has atrophic effect on the oxyntic gland and colon mucosa (Johnson 1981), as well as on certain carcinomas of the colon. Gastrin-receptor-blocking agents inhibit the growth of these gastrin-sensitive carcinomas (Imdahl et al. 1989; Eggstein and Imdahl 1988). Because the trophic effect of gastrin is at least partially inhibited
38 b y D F M O (Seidel et al. 1985), a c o n n e c t i o n with p o l y a m i n e m e t a b o l i s m is suggested. A t least in the colon, no i n d u c t i o n o f O D C b y gastrin has been i n d i c a t e d (Seidel et al. 1985; F i t z p a t r i c k et al. 1987). Recently, J o h n s o n et al. (1988) f o u n d i n c r e a s e d c o n c e n t r a t i o n s o f O D C in a s u b p o p u l a t i o n o f r a t o x y n t i c g l a n d cells after t r o p h i c s t i m u l a t i o n with gastrin, using a n t i s e r u m - d i l u t i o n techniques. I n v e s t i g a t i o n s c o n c e r n i n g the influence o f g a s t r i n o n O D C activity in c o l o n i c c a r c i n o m a o r h u m a n c o l o n m u c o s a h a v e n o t yet been p u b l i s h e d . P r o v e d k n o w l e d g e o f a p o s s i b l e influence o f gastrin o r g a s t r i n - r e c e p t o r - b l o c k ing agents o n O D C activity in c o l o n c a n c e r c o u l d shed new light o n t h e r a p y . I n this r e p o r t we p r e s e n t results o n the influence o f gastrin, e p i d e r m a l g r o w t h f a c t o r ( E G F ) , the gastrin rec e p t o r b l o c k e r b e n z o t r y p t e a n d D F M O o n the O D C activity a n d the g r o w t h o f f o u r h u m a n c o l o n i c c a r c i n o m a cell lines in vitro a n d p a r t l y in x e n o t r a n s p l a n t s o n n u d e mice. O u r results s h o w t h a t O D C activity is s t i m u l a t e d b y g a s t r i n a n d gastrin r e c e p t o r b l o c k e r s in the gastrin-sensitive c o l o n c a r c i n o m a cell line S W 403 in vitro a n d in the n u d e mice m o d e l . O D C activity a n d g r o w t h o f all cell lines were i n h i b i t e d b y D F M O .
Materials and methods Cell lines. The human colonic-carcinoma-derived cell lines SW 403, LS 174 T, Lovo and SW 1116 of the American Type Culture Collection (kindly supplied by the lnstitnt ffir Immunbiologie, Freiburg, FRG) were cultured in Dulbecco's or RPM I medium with 10 % fetal calf serum and antibiotics in 10-cm petri dishes containing 10 ml medium at 37~ C, 95% air and 5% CO2. Medium was replaced every 2nd or 3rd day. Cells were in each case subcultivated before growing confluently. After trypsination (0.03% trypsin in Hank's EDTA, 30 min, 37 ~ C) and eosin staining for viability 7 x 104 3 x 105 cells/ml were seeded. Cells were counted in a Neubauer cytometer. Cell culture materials were purchased from Gibco, USA. The following substances were added to the media (without preincubation): 10 ~tg/ml pentagastrin (Gastrodiagnost, Merck, Darmstadt, FRG), 8 Ixg/ml benzotrypte (kindly donated by Dr. Rovati, Rotta Research, Milano, italy), 8 ~tg/ml DFMO (kindly donated by Merell Dow Research, StraBburg, France), benzotrypte with DFMO in the concentrations mentioned above, 5 ng/ml EGF (Sigma, St. Louis, USA). Cell number and ODC were determined after 3 days and 8 days. In order to investigate the influence of medium replacement on ODC activity, SW 403 cells were preincubated over 48 h, the medium was replaced, and ODC was determined after the periods described below. Nude mice experiments. Experiments were carried out as already described (Imdahl et al. 1989): 105, SW 403 and LS 174 T cells were injected subcutaneously. The developing tumours were pooled and assayed for ODC activity after 4 weeks of treatment with 60 gg pentagastrin and 100 ~tg proglumide (kindly donated by Dr. Rovati, Rotta Research, Milano, Italy) and 0.9% NaC1 as control injected twice daily intraperitoneally. Determination of ODC activity. The assay was modified according to Bauknecht et al. (1987). All procedures were carried out in ice. Tumours were immediately frozen in liquid nitrogen and stored at - 7 0 ~ C. Cells were washed in 0.9% NaC1 after trypsination and the 200g (4 ~ C, 10 min) pellet was frozen. After homogenisation with the Ultratur-
rax homogeniser (IKA, Staufen, FRG) in 1.5 ml/g extraction buffer [0.1 M HEPES, 9 mM dithioerithritol (Aldrich, Steinheim, FRG), 5.0 mM EDTA (Merck, Darmstadt, FRG), 1.2 mM pyridoxal 5'phosphate (Sigma, St. Louis, USA), pH 6,8] three samples of the 35000 g supernatant (4~ C, 20 rain) were assayed for ODC and protein, by the method of Lowry et al. (1951). Assay tubes containing 400 gl supernatant, 390 gl extraction buffer and 10 nmol [xgCOz]ornithine (Amersham, Buckinghamshire, England) in 10 ~tl aqueous solution with 2% ethanol were placed in scintillation vials with 2 ml phenylethylamine (Aldrich, Steinheim, FRG), capped, and incubated 60 rain at 37~ C. The reaction was terminated and the liberated 14CO2 expelled by adding 250 ~tl 20% trichloroacetic acid by means of an injection cannula through the closed cap. After another 2 h incubation the reaction tubes were removed, and the scintillation vials were filled with 10 ml scintillation fluid (Rotiszint, Roth, Karlsruhe, FRG). The liberated 14CO2 was estimated in a liquid scintillation counter (Rackbeta, LKB, Bromma, Sweden). The activity of ODC was assayed as liberated 1~CO2 in pmol mg protein-1 h - t as a mean value of three samples. For statistics the Student t-test was used.
Results S W 403 cells
T h e g r o w t h o f S W 403 cells was significantly ( P < 0 . 0 1 ) s t i m u l a t e d b y p e n t a g a s t r i n (Table 1). O n the 3rd d a y the g a s t r i n - s t i m u l a t e d cells h a d m u l t i p l i e d five times, w h e r e a s the c o n t r o l cells h a d m u l t i p l i e d f o u r times only. 'The O D C activity i n c r e a s e d f r o m 25 p m o l h - 1 m g - 1 in c o n t r o l to 50 p m o l h -1 m g -1 w h e n p e n t a g a s t r i n was added. T h e g a s t r i n r e c e p t o r b l o c k e r b e n z o t r y p t e significantly i n h i b i t e d the g r o w t h o f S W 403 cells ( P < 0 . 0 1 ) ; the cell p r o l i f e r a t i o n was r e d u c e d to 80% o f c o n t r o l after 3 days. C o m p a r e d w i t h the c o n t r o l , a p p l i c a t i o n o f b e n z o t r y p t e resulted in the d o u b l i n g o f O D C activity after 3 days. D F M O r e d u c e d the O D C activity to 6 0 % o f control. Cell g r o w t h was r e d u c e d to the s a m e degree after 3 days. C o m b i n a t i o n o f D F M O a n d b e n z o t r y p t e h a d the s a m e effect as the a d d i t i o n o f one s u b s t a n c e alone. A p p l i c a t i o n o f E G F resulted in a m a r k e d increase o f cell g r o w t h (Table 2). W e o b s e r v e d a 3 0 % increase o f
Table 1. Influence of pentagastrin, benzotrypte, difluoromethylornithine (DFMO) and benzotrypte together with DFMO on ornithine decarboxylase (ODC) activity and growth of SW 403 cells" Treatment
10 5 x Cell growth by day 3 (cells/ml)
ODC activity on day 3 (pmol h - 1 mg protein- 1)
Control Pentagastrin (10 gg/ml) Benzotrypte (8 gg/ml) DFMO (8 ~tg/ml) Benzotrypte + DFMO
5.5 __0.2 6.7 • 0.2 *
25 • 2.3 50 + 0.4 *
4.4 • 0.4 *
42 _+5.4 *
3.6 + 0.2 *
15 + 0.8 *
3.8•
i1 -I-0.3 *
*
" Values represent means + standard deviation of four experiments. Each group contained six culture plates. Initial concentration of cells on day 0 = 1.33 x 105 cells/ml * Significant difference (P
