Cell Biology International Reports, VoL 16, No. 2, 1992
145
HUMAN HEPATOCYTE GROWTH FACTOR STIMULATES THE GROWTH OF HUH-6 CLONE 5 HUMAN H E P A T O B L A S T O M A CELLS Masahiro Miyazaki*, Eiichi Gohda 2. ", So Tsuboi*, Hirohito Tsubouchi 3 , Yasushi Daikuhara 4 , Masayoshi Namba l and Itaru Yamamoto 2 ~Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama U n i v e r s i t y Medical School, Okayama 700, Japan 2Department of Immunochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Japan 3Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan 4Department of Biochemistry, Kagoshima University Dental School, Kagoshima 890, Japan
ABSTRACT The effects of h u m a n hepatocyte growth factor (hHGF), a potent m i t o g e n for rat and human hepatoc~rtes in primary culture, on p r o l i f e r a t i o n of human hepatoma and hepatoblastoma cells were examined. Out of five cell lines; HLE, HUH-6 Clone 5, HUH-7, PLC/PRF/5, and Hep G2, only HUH-6 Clone 5 cells were stimulated by recombinant hHGF. Both native and recombinant hHGFs caused dose-dependent increases in cell number and DNA synthesis of cells. This stimulation was strongly inhibited by anti-hHGF monoclonal antibody. INTRODUCTION A c t i v i t y of hepatocyte growth factor (HGF), which stimulates DNA synthesis in rat hepatoc!rtes in primary culture, was detected in serum of normal and h e p a t e c t o m i z e d rats (Strain et al., 1982; M i c h a l o p o u l o s et al., 1982; Nakamura et al., 1984), rat platelet extracts (Russell et al., 1984; Paul and Piasecki, 1984) and human serum (Selden et al., 1986). We found a marked increase of this activity in the serum and plasma of patients with fulminant hepatic failure (Nakayama et al., 1985). We purified human h e p a t o c y t e growth factor (hHGF) from the plasma of these patients and reported that this factor is composed of two peptide chains with molecular weights of 54,000-65,000 and 31,500-34,500, linked together probably by a single d i s u l f i d e bond (Gohda et al., 1988). Nakamura et al. (1987) and Zarnegar and Michalopoulos (1989) also purified HGF from rat platelets,
*To w h o m c o r r e s p o n d e n c e
should be addressed.
0309-1651/92/020145-10/$03.00/0
© 1992 Academic Press Ltd
146
Cell Biology International Reports, Vol. 16, No. 2, 1992
and rabbit serum and normal human plasma, respectively. Our group (Miyazawa et al., 1989) and others (Nakamura et al., 1989; Rubln et al., 1991) recently d e d u c e d the complete p r i m a r y structure of hHGF from the nucleotide sequence of h H G F cDNA. These sequence data have revealed that hHGF is a new g r o w t h factor, which is synthesized as a single p o l y p e p t i d e c h a i n w i t h a signal peptide at the N-terminal. Evidence has a c c u m u l a t e d that supports the p o s s i b i l i t y that HGF acts as a h e p a t o t r o p h i c factor in liver regeneration. Levels of HGF and an h H G F - l i k e factor m a r k e d l y increased in serum and liver of mice and rats treated w i t h c a r b o n t e t r a c h l o r i d e prior to r e g e n e r a t i o n of the damaged liver (Gohda et al., 1990a; Asami et al., 1991). hHGF stimulates DNA synthesis of adult rat h e p a t o c y t e s in p r i m a r y culture at less t h a n one-tenth the m o l a r concentrations of epidermal growth factor (EGF) and transforming growth factor- G ( T G F - ~ ) , known as potent m i t o g e n s for h e p a t o c y t e s (Gohda et al., 1990b). H e p a t o c y t e s s t i m u l a t e d by hHGF not only enter the S-phase, but also divide w i t h o u t the addition of any other factors (Gohda et al., 1991). Thus, hHGF is the most potent g r o w t h factor for rat hepatoclrtes in p r i m a r y culture reported to date. Recently Strain et al. (1991) reported that human hepatocytes were m o r e responsive to hHGF than rat hepatocytes. These findings p r o m p t e d us to e x a m i n e w h e t h e r human h e p a t o m a cells are r e s p o n s i v e to hHGF or not. In this study, we used three human hepatoma-derived cell lines; HLE, HUH-7, and PLC/PRF/5, and two h e p a t o b l a s t o m a - d e r i v e d cell lines; HUH-6 Clone 5 and Hep G2. We found that hHGF s t i m u l a t e d p r o l i f e r a t i o n of HUH-6 Clone 5 cells and inhibited that of Hep G2 cells. M A T E R I A L S AND M E T H O D S Materials: Eagle's m i n i m u m essential m e d i u m (MEM) and RPMI 1640 m e d i u m were p u r c h a s e d from Nissui Pharmaceutical Co., Ltd., Tokyo; l a c t a l b u m i n h y d r o l y s a t e (LAH) was from Difco Laboratories, Detroit, MI; mouse EGF and b o v i n e insulin w e r e from Sigma Chemical Co., St.Louis, MO; and [methyl-3H]thymidine (0.74 TBq/mmol) was from Du Pont-New England Nuclear, Boston, MA. N a t i v e hHGF was purified from plasma of patients w i t h fulminant hepatic failure, which was obtained during plasma exchange therapy, as described previously (Gohda et al., 1988). R e c o m b i n a n t h H G F (rhHGF), which was p r o d u c e d in C h i n e s e h a m s t e r o v a r y cells t r a n s f e c t e d w i t h the hHGF cDNA and purified (Strain et al., 1991), was k i n d l y provided by the R e s e a r c h Center, Mitsubishi Kasei Corporation, Yokohama. Mouse anti-hHGF monoclonal antibody, purified partially from mouse ascitic fluid by MAPS kit II (Bio-Rad) (Tsubouchi et al., 1991), and an enzyme-linked immunosorbent assay (ELISA) kit for bHGF (Tsubouchi et al., 1991) were k i n d l y supplied by O t s u k a A s s a y Laboratories, O t s u k a Pharmaceutical Co., Ltd., Tokushima. HLE (Doi et al., 1975), HUH-6 Clone 5 (Doi, 1976), and HUH-7
Cell Biology International Reports, VoL 16, No. 2, 1992
147
(Nakabayashi et al., 1982) cell lines were established and m a i n t a i n e d in the I n s t i t u t e of M o l e c u l a r and C e l l u l a r Biology, O k a y a m a U n i v e r s i t y M e d i c a l School. PLC/PRF/5 ( A l e x a n d e r et al., 1976) and Hep G2 (Aden et al., 1979) c e l l lines w e r e o b t a i n e d f r o m J C R B and ATCC, r e s p e c t i v e l y . Culture of human hepatoma and hepatoblastoma cells: Stock c u l t u r e s of of HLE, H U H - 6 C l o n e 5, HUH-7, PLC/PRF/5, and H e p G2 c e l l s w e r e g r o w n as m o n o l a y e r s in M E M c o n t a i n i n g 20% b o v i n e s e r u m (BS), RPMI 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 2 . 5 % BS, R P M I 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 1% fetal b o v i n e s e r u m (FBS), RPMI 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 2.5% FBS, and RPMI 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 10% FBS, r e s p e c t i v e l y . The c u l t u r e s w e r e i n c u b a t e d at 37 ~ in a h u m i d i f i e d a t m o s p h e r e of 5% C02 in air. For the experiments, the hepatoma and hepatoblastoma cells were trypsinized, suspended in t h e s a m e m e d i a as t h o s e for s t o c k cultures, and s e e d e d in 2 4 - w e l l d i s h e s (Falcon) c o a t e d w i t h rat tail c o l l a g e n at a d e n s i t y of 5 X 104 c e l l s / 0 . 5 m l / w e l l (HLE, HUH-7, PLC/PRF/5, a n d Hep G2) or 5 X 104 nuclei/0.5 ml/well (HUH-6 C l o n e 5). The m e d i u m was replaced w i t h M E M c o n t a i n i n g 2 . 5 % BS (HLE), R P M I 1 6 4 0 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 1% FBS (HUH-7, P L C / P R F / 5 , a n d Hep G2), o r R P M I 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 1% BS (HUH-6 C l o n e 5), 24 h after seeding to r e m o v e u n a t t a c h e d cells, and rhHGF, native hHGF, and o t h e r g r o w t h f a c t o r s w e r e a d d e d to the c u l t u r e s . The culture medium was renewed every 2 days thereafter and the growth factors were added at each time. Cell growth was m e a s u r e d b y c o u n t i n g v i a b l e c e l l s (HLE, HUH-7, PLC/PRF/5, and H e p G2), e x c l u d i n g t r y p a n blue, or n u c l e i (HUH-6 C l o n e 5), in a hemocytometer. The statistical significance was analyzed by Student's t-test Primary Culture of hepatocytes from adult rat liver: Hepatocytes were prepared from adult male Wistar rats, w e i g h i n g a b o u t 200 g, and w e r e c u l t u r e d as d e s c r i b e d p r e v i o u s l y ( G o h d a et al., 1988). The c u l t u r e m e d i u m w a s W i l l i a m s m e d i u m E, supplemented with 5% FBS, i0 nM dexamethasone, i00 U/ml p e n i c i l l i n , and i00 ~ g / m l s t r e p t o m y c i n . D e t e r m i n a t i o n of D N A s y n t h e s i s : D N A s y n t h e s i s in h e p a t o m a and h e p a t o b l a s t o m a c e l l s or in rat h e p a t o c y t e s was d e t e r m i n e d b y l a b e l i n g the c e l l s w i t h [ 3 H ] t h y m i d i n e (92 kBq/ml, 46 G B q / m m o l ) for 41 h at 3 7 ~ , b e t w e e n 6 and 47 h a f t e r the f i r s t a d d i t i o n of growth factors, or with [aH]thymidine (74 kBq/ml, 37 G B q / m m o l ) for 18 h at 37~C b e t w e e n 29 and 47 h a f t e r s e e d i n g in the p r e s e n c e o r a b s e n c e of i0 m M h y d r o x y u r e a , and w a s e x p r e s s e d as i n c o r p o r a t e d [ 3 H ] t h y m i d i n e p e r N g of c e l l u l a r p r o t e i n , as described previously ( M i y a z a k i et al., 1990; G o h d a et al., 1988). P r o t e i n w a s d e t e r m i n e d b y the m e t h o d of L o w r y et al. (1951). hHGF
ELISA:
The
sandwich
ELISA
for
hHGF
was
performed
as
Cell Biology lnternational Reports, VoL 16, No. 2, 1992
148 described RESULTS
previously
(Tsubouchi
et al.,
1991).
AND DISCUSSION
Table 1 shows the effects of r h H G F on proliferation of HLE, H U H - 6 C l o n e 5, HUH-7, PLC/PRF/5, and H e p G2 cells. A s r e p o r t e d previously (Strain et al., 1991), this rhHGF stimulated DNA synthesis in human hepatoclrtes in primary culture at doses
Table Effects
Cell
1
of r h H G F on p r o l i f e r a t i o n of HLE, H U H - 6 HUH-7, PLC/PRF/5, a n d H e p G2 c e l l s
line
rhHGF (ng/ml)
Cell Day
HLE HUH-6
-
Clone
HUH-7
PLC/PRF/5 Hep G2
5
30 30 30 3O 30
1
4.9+-0.i 3.4+- 0.2 2.7+- 0.3 3.6+- 0.3 6.2+- 0.6
Clone
5,
number a (× 104/well) Day 4 11.6+-0.2 11.4+-0.9 9.6+- 0.8 16.2+- 0.9 ~ 7.6+__ 0.6 6.7+- 0.2 14.9+- 1.0 14.9+- 0.8 31.6+- 0.9 24.0+- 0.7 b
Day 7 20.4+-0.8 20.9+-1.5 29.5+- 0.7 45.2+- 1.2 b 18.7+- 0.6 17.3+- 0.3 27.0+- 1.4 27.3+- 1.2
r h H G F w a s a d d e d t o the c u l t u r e s o n d a y 1 (24 h a f t e r s e e d i n g ) and t h e r e a f t e r at e a c h m e d i u m c h a n g e . Values a r e mean+- S.D. for t r i p l i c a t e dishes. aFor H U H - 6 C l o n e 5 cells, n u c l e u s n u m b e r was c o u n t e d . Values that a r e s i g n i f i c a n t l y different from t h a t of c u l t u r e s w i t h o u t r h H G F a r e i n d i c a t e d by:
bp
145
HUMAN HEPATOCYTE GROWTH FACTOR STIMULATES THE GROWTH OF HUH-6 CLONE 5 HUMAN H E P A T O B L A S T O M A CELLS Masahiro Miyazaki*, Eiichi Gohda 2. ", So Tsuboi*, Hirohito Tsubouchi 3 , Yasushi Daikuhara 4 , Masayoshi Namba l and Itaru Yamamoto 2 ~Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama U n i v e r s i t y Medical School, Okayama 700, Japan 2Department of Immunochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Japan 3Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan 4Department of Biochemistry, Kagoshima University Dental School, Kagoshima 890, Japan
ABSTRACT The effects of h u m a n hepatocyte growth factor (hHGF), a potent m i t o g e n for rat and human hepatoc~rtes in primary culture, on p r o l i f e r a t i o n of human hepatoma and hepatoblastoma cells were examined. Out of five cell lines; HLE, HUH-6 Clone 5, HUH-7, PLC/PRF/5, and Hep G2, only HUH-6 Clone 5 cells were stimulated by recombinant hHGF. Both native and recombinant hHGFs caused dose-dependent increases in cell number and DNA synthesis of cells. This stimulation was strongly inhibited by anti-hHGF monoclonal antibody. INTRODUCTION A c t i v i t y of hepatocyte growth factor (HGF), which stimulates DNA synthesis in rat hepatoc!rtes in primary culture, was detected in serum of normal and h e p a t e c t o m i z e d rats (Strain et al., 1982; M i c h a l o p o u l o s et al., 1982; Nakamura et al., 1984), rat platelet extracts (Russell et al., 1984; Paul and Piasecki, 1984) and human serum (Selden et al., 1986). We found a marked increase of this activity in the serum and plasma of patients with fulminant hepatic failure (Nakayama et al., 1985). We purified human h e p a t o c y t e growth factor (hHGF) from the plasma of these patients and reported that this factor is composed of two peptide chains with molecular weights of 54,000-65,000 and 31,500-34,500, linked together probably by a single d i s u l f i d e bond (Gohda et al., 1988). Nakamura et al. (1987) and Zarnegar and Michalopoulos (1989) also purified HGF from rat platelets,
*To w h o m c o r r e s p o n d e n c e
should be addressed.
0309-1651/92/020145-10/$03.00/0
© 1992 Academic Press Ltd
146
Cell Biology International Reports, Vol. 16, No. 2, 1992
and rabbit serum and normal human plasma, respectively. Our group (Miyazawa et al., 1989) and others (Nakamura et al., 1989; Rubln et al., 1991) recently d e d u c e d the complete p r i m a r y structure of hHGF from the nucleotide sequence of h H G F cDNA. These sequence data have revealed that hHGF is a new g r o w t h factor, which is synthesized as a single p o l y p e p t i d e c h a i n w i t h a signal peptide at the N-terminal. Evidence has a c c u m u l a t e d that supports the p o s s i b i l i t y that HGF acts as a h e p a t o t r o p h i c factor in liver regeneration. Levels of HGF and an h H G F - l i k e factor m a r k e d l y increased in serum and liver of mice and rats treated w i t h c a r b o n t e t r a c h l o r i d e prior to r e g e n e r a t i o n of the damaged liver (Gohda et al., 1990a; Asami et al., 1991). hHGF stimulates DNA synthesis of adult rat h e p a t o c y t e s in p r i m a r y culture at less t h a n one-tenth the m o l a r concentrations of epidermal growth factor (EGF) and transforming growth factor- G ( T G F - ~ ) , known as potent m i t o g e n s for h e p a t o c y t e s (Gohda et al., 1990b). H e p a t o c y t e s s t i m u l a t e d by hHGF not only enter the S-phase, but also divide w i t h o u t the addition of any other factors (Gohda et al., 1991). Thus, hHGF is the most potent g r o w t h factor for rat hepatoclrtes in p r i m a r y culture reported to date. Recently Strain et al. (1991) reported that human hepatocytes were m o r e responsive to hHGF than rat hepatocytes. These findings p r o m p t e d us to e x a m i n e w h e t h e r human h e p a t o m a cells are r e s p o n s i v e to hHGF or not. In this study, we used three human hepatoma-derived cell lines; HLE, HUH-7, and PLC/PRF/5, and two h e p a t o b l a s t o m a - d e r i v e d cell lines; HUH-6 Clone 5 and Hep G2. We found that hHGF s t i m u l a t e d p r o l i f e r a t i o n of HUH-6 Clone 5 cells and inhibited that of Hep G2 cells. M A T E R I A L S AND M E T H O D S Materials: Eagle's m i n i m u m essential m e d i u m (MEM) and RPMI 1640 m e d i u m were p u r c h a s e d from Nissui Pharmaceutical Co., Ltd., Tokyo; l a c t a l b u m i n h y d r o l y s a t e (LAH) was from Difco Laboratories, Detroit, MI; mouse EGF and b o v i n e insulin w e r e from Sigma Chemical Co., St.Louis, MO; and [methyl-3H]thymidine (0.74 TBq/mmol) was from Du Pont-New England Nuclear, Boston, MA. N a t i v e hHGF was purified from plasma of patients w i t h fulminant hepatic failure, which was obtained during plasma exchange therapy, as described previously (Gohda et al., 1988). R e c o m b i n a n t h H G F (rhHGF), which was p r o d u c e d in C h i n e s e h a m s t e r o v a r y cells t r a n s f e c t e d w i t h the hHGF cDNA and purified (Strain et al., 1991), was k i n d l y provided by the R e s e a r c h Center, Mitsubishi Kasei Corporation, Yokohama. Mouse anti-hHGF monoclonal antibody, purified partially from mouse ascitic fluid by MAPS kit II (Bio-Rad) (Tsubouchi et al., 1991), and an enzyme-linked immunosorbent assay (ELISA) kit for bHGF (Tsubouchi et al., 1991) were k i n d l y supplied by O t s u k a A s s a y Laboratories, O t s u k a Pharmaceutical Co., Ltd., Tokushima. HLE (Doi et al., 1975), HUH-6 Clone 5 (Doi, 1976), and HUH-7
Cell Biology International Reports, VoL 16, No. 2, 1992
147
(Nakabayashi et al., 1982) cell lines were established and m a i n t a i n e d in the I n s t i t u t e of M o l e c u l a r and C e l l u l a r Biology, O k a y a m a U n i v e r s i t y M e d i c a l School. PLC/PRF/5 ( A l e x a n d e r et al., 1976) and Hep G2 (Aden et al., 1979) c e l l lines w e r e o b t a i n e d f r o m J C R B and ATCC, r e s p e c t i v e l y . Culture of human hepatoma and hepatoblastoma cells: Stock c u l t u r e s of of HLE, H U H - 6 C l o n e 5, HUH-7, PLC/PRF/5, and H e p G2 c e l l s w e r e g r o w n as m o n o l a y e r s in M E M c o n t a i n i n g 20% b o v i n e s e r u m (BS), RPMI 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 2 . 5 % BS, R P M I 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 1% fetal b o v i n e s e r u m (FBS), RPMI 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 2.5% FBS, and RPMI 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 10% FBS, r e s p e c t i v e l y . The c u l t u r e s w e r e i n c u b a t e d at 37 ~ in a h u m i d i f i e d a t m o s p h e r e of 5% C02 in air. For the experiments, the hepatoma and hepatoblastoma cells were trypsinized, suspended in t h e s a m e m e d i a as t h o s e for s t o c k cultures, and s e e d e d in 2 4 - w e l l d i s h e s (Falcon) c o a t e d w i t h rat tail c o l l a g e n at a d e n s i t y of 5 X 104 c e l l s / 0 . 5 m l / w e l l (HLE, HUH-7, PLC/PRF/5, a n d Hep G2) or 5 X 104 nuclei/0.5 ml/well (HUH-6 C l o n e 5). The m e d i u m was replaced w i t h M E M c o n t a i n i n g 2 . 5 % BS (HLE), R P M I 1 6 4 0 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 1% FBS (HUH-7, P L C / P R F / 5 , a n d Hep G2), o r R P M I 1640 m e d i u m c o n t a i n i n g 0 . 2 % LAH and 1% BS (HUH-6 C l o n e 5), 24 h after seeding to r e m o v e u n a t t a c h e d cells, and rhHGF, native hHGF, and o t h e r g r o w t h f a c t o r s w e r e a d d e d to the c u l t u r e s . The culture medium was renewed every 2 days thereafter and the growth factors were added at each time. Cell growth was m e a s u r e d b y c o u n t i n g v i a b l e c e l l s (HLE, HUH-7, PLC/PRF/5, and H e p G2), e x c l u d i n g t r y p a n blue, or n u c l e i (HUH-6 C l o n e 5), in a hemocytometer. The statistical significance was analyzed by Student's t-test Primary Culture of hepatocytes from adult rat liver: Hepatocytes were prepared from adult male Wistar rats, w e i g h i n g a b o u t 200 g, and w e r e c u l t u r e d as d e s c r i b e d p r e v i o u s l y ( G o h d a et al., 1988). The c u l t u r e m e d i u m w a s W i l l i a m s m e d i u m E, supplemented with 5% FBS, i0 nM dexamethasone, i00 U/ml p e n i c i l l i n , and i00 ~ g / m l s t r e p t o m y c i n . D e t e r m i n a t i o n of D N A s y n t h e s i s : D N A s y n t h e s i s in h e p a t o m a and h e p a t o b l a s t o m a c e l l s or in rat h e p a t o c y t e s was d e t e r m i n e d b y l a b e l i n g the c e l l s w i t h [ 3 H ] t h y m i d i n e (92 kBq/ml, 46 G B q / m m o l ) for 41 h at 3 7 ~ , b e t w e e n 6 and 47 h a f t e r the f i r s t a d d i t i o n of growth factors, or with [aH]thymidine (74 kBq/ml, 37 G B q / m m o l ) for 18 h at 37~C b e t w e e n 29 and 47 h a f t e r s e e d i n g in the p r e s e n c e o r a b s e n c e of i0 m M h y d r o x y u r e a , and w a s e x p r e s s e d as i n c o r p o r a t e d [ 3 H ] t h y m i d i n e p e r N g of c e l l u l a r p r o t e i n , as described previously ( M i y a z a k i et al., 1990; G o h d a et al., 1988). P r o t e i n w a s d e t e r m i n e d b y the m e t h o d of L o w r y et al. (1951). hHGF
ELISA:
The
sandwich
ELISA
for
hHGF
was
performed
as
Cell Biology lnternational Reports, VoL 16, No. 2, 1992
148 described RESULTS
previously
(Tsubouchi
et al.,
1991).
AND DISCUSSION
Table 1 shows the effects of r h H G F on proliferation of HLE, H U H - 6 C l o n e 5, HUH-7, PLC/PRF/5, and H e p G2 cells. A s r e p o r t e d previously (Strain et al., 1991), this rhHGF stimulated DNA synthesis in human hepatoclrtes in primary culture at doses
Table Effects
Cell
1
of r h H G F on p r o l i f e r a t i o n of HLE, H U H - 6 HUH-7, PLC/PRF/5, a n d H e p G2 c e l l s
line
rhHGF (ng/ml)
Cell Day
HLE HUH-6
-
Clone
HUH-7
PLC/PRF/5 Hep G2
5
30 30 30 3O 30
1
4.9+-0.i 3.4+- 0.2 2.7+- 0.3 3.6+- 0.3 6.2+- 0.6
Clone
5,
number a (× 104/well) Day 4 11.6+-0.2 11.4+-0.9 9.6+- 0.8 16.2+- 0.9 ~ 7.6+__ 0.6 6.7+- 0.2 14.9+- 1.0 14.9+- 0.8 31.6+- 0.9 24.0+- 0.7 b
Day 7 20.4+-0.8 20.9+-1.5 29.5+- 0.7 45.2+- 1.2 b 18.7+- 0.6 17.3+- 0.3 27.0+- 1.4 27.3+- 1.2
r h H G F w a s a d d e d t o the c u l t u r e s o n d a y 1 (24 h a f t e r s e e d i n g ) and t h e r e a f t e r at e a c h m e d i u m c h a n g e . Values a r e mean+- S.D. for t r i p l i c a t e dishes. aFor H U H - 6 C l o n e 5 cells, n u c l e u s n u m b e r was c o u n t e d . Values that a r e s i g n i f i c a n t l y different from t h a t of c u l t u r e s w i t h o u t r h H G F a r e i n d i c a t e d by:
bp
