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Photoreactive epidermal growth factor (EGF) was synthesized by
conjugating mouse EGF with photoreactive polyallylamine, which
was synthesized by the coupling reaction of polyallylamine with
N-(4-azidobenzoyloxy)succinimide. The EGF derivative was
pattern-immobilized onto a polystyrene plate by UV-irradiation in
the presence of a photomask in a prescribed micro-pattern. The
patterned immobilization of EGF on the polystyrene plate was
confirmed by immunostaining with anti-EGF antibody. Chinese
hamster ovary (CHO) cells overexpressing EGF receptors were
cultured on the micro-patterned plate. The phosphorylated
tyrosine residues of EGF receptors and signal proteins were
detected only in the cells adhered in the EGF-immobilized area
and cell growth was observed only in the EGF-immobilized area.
The cells growing in the EGF-immobilized area were partially
stained by anti-phosphotyrosine antibody, when the area of EGF
immobilization was smaller than the cell. The partial staining of
activated proteins indicates that immobilization of EGF inhibited
the free lateral diffusion and internalization of the activated
EGF/EGF-receptor complex. The enhanced cell growth is due to
juxtacrine stimulation realized by immobilized EGF.
Natural and artificial substrata are important in basic
bioscience and biotechnology including cell culture and tissue
engineering (Mrksich et al., 1996; Peppas and Langer, 1994;
Hubbell, 1995; Gumbiner and Yamada, 1995). Cellular interactions
with the extracellular matrix play critical roles in various
biological processes, including migration, morphogenesis, growth,
differentiation and apoptosis (Roskelly et al, 1995). On the
other hand, selective cell attachment (Lee et al, 1993; Spargo et
al, 1994; Connolly, 1994; Clemence et al, 1995) and cell shape
regulation (Singhvi et al, 1994; Chen et al, 1997) by
micro-patterned surface substrata constructed by nanotechnology
have been reported. However, those artificial substrata could not
transduce biological signals such as for growth and
differentiation. Recently, several growth factors including the
epidermal growth factor (EGF) have been reported to regulate cell
functions in the transmembrane form by juxtacrine stimulation
(Massague and Pandiella, 1993; Higashiyama et al 1995). They
stimulate cells without internalization. The juxtacrine mechanism
was deduced from the studies of intercellular regulation by
paraformaldehyde-fixed cells that express the growth factors
(Higashiyama et al 1995).
In addition to the juxtacrine stimulation, recently some
researchers including our group demonstrated artificial
juxtacrine stimulation by interleukin 2 or insulin immobilized on
artificial substrata such as polystyrene and poly(methyl
methacrylate) films (Horwitz et al, 1993; Ito et al, 1996; Chen
et al, 1997). In the present investigation, photoreactive EGF
conjugate was synthesized and immobilized on a prescribed
micro-pattern of different size to visualize signal transduction
without internalization. EGF transduced the signal through the
cognate receptor to stimulate the growth of Chinese hamster ovary
cells overexpressing EGF receptors (CHO-ER cells).
Materials and Methods
N,N-dimethylformamide (DMF), Paraformaldehyde, triton X-100
and sodium orthovanadate were purchased from Wako Pure Chem. Ltd.
(Osaka, Japan). Dicyclohexylcarbodiimide (DCC) and 4-azidobenzoic
acid were purchased from Tokyo Kasei Co. (Tokyo, Japan).
N-hydroxysuccinimide was purchased from the Protein Institute
Inc. (Minoh, Japan). Polyallylamine hydrochloride was purchased
from Nittobo (Tokyo, Japan). Bovine serum albumin (BSA) was
purchased from Intergen Co. (Purchase, NY). Mouse EGF was
purchased from Toyobo (Osaka, Japan). Anti-EGF IgG was purchased
from Becton Dickinson Labware (Bedford, MA). Anti-phosphotyrosine
antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Rhodamine conjugated antibody was purchased from
Protos Immunoresearch (San Francisco, CA). VectashieldTM mounting
medium for fluorescence was purchased from Vector Laboratories,
Inc. (Burlingame, CA). Tissue culture polystyrene plates with 6
wells (Sumilon) were purchased from Akita Sumitomo Bake Co.
Preparation of photoreactive polyallylamine and photoreactive
Photoreactive EGF was synthesized by conjugating mouse EGF
with the photoreactive polyallylamine, which was synthesized by
the coupling reaction of polyallylamine with
N-(4-azidobenzoyloxy)succinimide was prepared as described by
Matsuda and Sugawara (1995). A solution of DCC (13.3 g, 64.6
mmol) in tetrahydrofuran (THF, 50 mL) was added dropwise to a
solution of N-hydroxysuccinimide (7.43 g, 64.6 mmol) and
4-azidobenzoic acid (9.57 g, 58.7 mmol) in THF (150 mL), then
cooled in an ice bath under stirring. After 3 h, the reaction
mixture was warmed slowly to room temperature, then stirred
overnight. The white solid that formed was filtered off and the
solvent was removed under reduced pressure. The yellow residue
obtained was crystallized from isopropyl alcohol/diisopropyl
Polyallylamine (MW = 60,000, 30 mg) dissolved in 10mL
phosphate-buffered solution (pH = 7.0) was added to DMF solution
(20 mL) of N-(4-azidobenzoyloxy)succinimide (25.8 mg) under
stirring on ice. After incubation at 4 C for 24 h under stirring,
the solution was ultrafiltrated (Millipore MoleCut II, cut-off
below 10 kDa) and washed three times with 10mL distilled water.
The azidophenyl-derivatized polyallylamine was referred to as
AzPhPAAm. The amount of azidophenyl groups in the conjugate
calculated from the absorbance at 280nm was 65 moles/mole. The
azidophenyl-derivatized polyallylamine was further conjugated
with EGF as follows. In a 0.1 M
2-(N-morpholino)-ethanesulfate-buffered solution (MES, pH = 4.5,
10 mL), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (water-soluble carbodiimide, WSC, 10 mg) and EGF
(300 microgram) and AzPhPAAm (600 microgram) were added, and
allowed to react in an ice bath for 24 h under stirring. Then the
solution was ultrafiltrated (Millipore MoleCut II, cut-off below
10 kDa) and washed twice with 2 mL distilled water. The
photo-reactive EGF conjugate was referred to as AzPhPAAmEGF. The
amount of EGF in the AzPhPAAmEGF conjugate determined by
measuring the fluorescence intensity at 345 nm by excitation at
280 nm was 1.4 moles/mole.
Pattern-immobilization of EGF.
An aqueous solution of AzPhPAAm (200 microgram/mL, 200
microlitter) was eluted on a polystyrene plate in the shape of a
circle (diameter of 10 mm) and air-dried at room temperature.
Then the plate was UV-irradiated using a UV lamp (Koala, 100 W)
from a distance of 5 cm for 10 s. The plate was thoroughly washed
with diluted hydrochloric acid (pH = 3.0) until the absence of
released AzPhPAAm was confirmed by ultraviolet absorbance at 280
nm (7 days). Subsequently, the EGF conjugate (200 microgram/mL,
50 microlitter) was cast in a circular shape (diameter of 5 mm)
and air-dried at room temperature. The plate was covered with a
photomask of a specific pattern and irradiated with an
ultraviolet lamp from a distance of 5 cm for 10 s. Finally, the
plate was washed with PBS until the absence of released EGF was
confirmed by ultraviolet absorbance at 280 nm (7 days).
Immunostaininig of immobilized EGF.
The plates immobilized with EGF were immersed in PBS
containing 0.02% NaN3 and 3% bovine serum albumin (BSA) in an ice
bath for 24 h, and subsequently incubated with anti-EGF IgG
antibody diluted in PBS containing 0.02% NaN3 and 3% bovine serum
albumin (2 mg/mL) in an ice bethfor 12 h. After being washed with
PBS containing 0.02% NaN3, the plate was incubated in PBS
containing rhodamine-conjugated anti-mouse IgG antibody (2 mg/mL)
and 0.02% NaN3 in an ice bath for 12 h. The stained plate was
washed with PBS, briefly rinsed with distilled water, mounted in
Vectashield mounting medium and observed under a laser
fluorescene microscope (Olympus Co., Tokyo, Japan).
Cell culture and treatments for microscopic observation.
CHO-ER cells (2.5 x 105 receptor molecules per cell) were
subcultured in Ham F-12 medium containing 10% (v / v) fetal
bovine serum. After culturing in the absence of serum for 2 days,
cells were harvested by incubation or 10 min with PBS contianing
0.02% (w/v) ethylenediamine tetraacetic acid and pipetting. After
being washed twice with Ham F-12 medium, the cells were suspended
in Ham F-12 medium (1 x 106 cells/mL). The cell suspension was
added to 6-well tissue culture plates (0.2 mL/well) containing
the EGF-immobilized polystyrene plate that had been incubated in
the well in the presence of Ham F-12 medium (5 mL) for 2 h. The
cells were cultured for 48 h and observed with a phase-contrast
To investigate the tyrosine phosphorylation of signal
proteins, the cells were cultured for 30 min. Then, the cells
were fixed for 30 min in an ice bath with 3% paraformaldehyde in
PBS. The fixed cells were washed three times with PBS containing
1 mM Na3VO4. Subsequently, the cells were permeabilized with PBS
containing 0.25% Triton X-100 and 1 mM Na3VO4, and washed three
times with 50 mM Tris-HCl-buffered solution containing 150 mM
NaCl and 0.1% Triton X-100 (TBST, pH = 7.4) and 1 mM Na3VO4.
After an overnight incubation in an ice bath in TBST containing
3% BSA and 1 mM Na3VO4, the treated cells were incubated for 2 h
at room temperature with a solution of anti-phosphotyrosine mouse
IgG diluted to 1 / 100 fold with 50mM Tris-HCl (pH 7.4), 150 mM
NaCl, 0.01% Tween 20, 0.02% NaN3, 1 mM Na3VO4 (TBS) containing 3%
BSA. The cells were washed once with TBS, once with TBST and once
with TBST containing 0.1% BSA. A solution of rhodamine-conjugated
anti-mouse IgG antibody was diluted to 1 / 200 fold with TBS
containing 3% BSA and incubated with the cells for 2 h at room
temperature. The cells were washed three times for 5 min each
with TBST, and three times with PBS, and briefly rinsed with
distilled water, and then mounted in Vectashield mounting medium.
The cells were observed by a laser fluorescence microscope.
Preparation of photoreactive EGF conjugate and
pattern-immobilization of EGF.
Photoreactive polyallylamine was synthesized by coupling
polyallylamine with N-(4-azidobenzoyloxy)succinimide. The amount
of incorporated azidophenyl groups in the conjugate calculated
from the absorbance at 280nm was 65 moles/mole. The
azidophenyl-derivatized polyallylamine was further conjugated
with EGF to synthesize the photoreactive EGF conjugate. The
content of EGF in the AzPhPAAmEGF conjugate determined by
measuring the fluorescence intensity at 345 nm by excitation at
280 nm was 1.4 moles/mole. A polystyrene plate was in advance
grafted with the photo-reactive polyallylamine by UV-irradiation.
Then, an aqueous solution of the photo-reactive EGF was coated on
the polyallylamine grafted polystyrene plate, air-dried, and the
plate was UV-irradiated in the presence of a photomask in a
prescribed micro-pattern. Upon UV irradiation, the azidophenyl
groups were easily photolyzed to generate highly reactive
nitrenes, which spontaneously formed covalent bonds with
neighboring hydrocarbons in the polystyrene plate surface. The
irradiated areas should be covalently cross-linked with EGF. The
photo-reactive EGF in other areas should not be crosslinked and
could be removed by washing. A pattern of covalently cross-linked
EGF would appear on the polystyrene plate surface.
After the plate was completely washed with PBS, the patterned
immobilization of EGF on the polystyrene plate was confirmed by
staining with anti-EGF antibody. The pattern of immobilized EGF
was the same as that of photomask used.
Tyrosine phosphorylation of signal proteins.
CHO-ER cells were cultured on the plate pattern-immobilized
with EGF for 30 min. The CHO-ER cells were fixed by
paraformaldehyde, and stained with anti-phosphotyrosine mouse IgG
and rhodamine-conjugated anti-mouse IgG antibodies. The uniform
adherence of cells on the surface of the plate
pattern-immobilized with EGF indicated that cell adhesion was not
enhanced by immobilized EGF. However, it was shown that the
phosphorylated tyrosine residue was detected only in the cells
adhered on the EGF-immobilized area, indicating a signal
transduction occurring only through immobilized EGF.
The cells adhered on the plate pattern-immobilized with EGF in
a narrow stripe pattern. The contact area (stripes of
2-micrometer in width) between the cells and the immobilized EGF
was stained by anti-phosphotyrosine antibody. Since the free
lateral diffusion and internalization of bound EGF/EGFR complex
are prohibited by immobilization of EGF, signal proteins were
partially activated. These findings indicate that the biological
signal is transduced only to the cells that interact with
When the width of the stripe is larger (100 micrometer) than
the cell, the patterned growth of CHO-ER cells was observed. The
patterned growth may have been caused by the enhancement of cell
growth due to signal transduction from the immobilized EGF. When
the pattern width is smaller than the cell (2 micrometer), all
the cells proliferated and patterned growth did not occur.
Considering the absence of cell growth in the EGF-non-immobilized
area, the partial and lateral diffusion-limited stimulation was
sufficient for the cell growth.
Although the conjugation of biosignal with an insoluble
substratum is an important technique for elucidation of the
biosignaling mechanism and molecular design of drugs or medical
materials, it has not sufficiently been examined after the
pioneering investigation using sepharose gel with immobilized
insulin (Cuatrecasas, 1969; Venter, 1982). The stimulation of the
growth of only the cells on the immobilized EGF region indicated
that diffusible EGF was absent in the culture system. The local
concentration of EGF on the surface is sufficient to promote
effective interaction with the EGF receptors of adsorbed cells.
Recent studies on overlapping of adhesion molecules and growth
factors (Clark and Brugge, 1995), the growth stimulation by
noninternalizing EGF receptors (Wells et al, 1990), and various
juxtacrine stimulators (Massague and Pandiella, 1993; Higashiyama
et al, 1995), suggest that, the signal of immobilized EGF was
transduced and cell growth stimulated without internalization. By
similar methods it will be possible to manipulate cells and
tissues using artificial substrata with covalently conjugated
growth factors and cytokines.
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