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Regulation of Cell Functions
by Micropattern-Immobilized
Biosignal Molecules

by
Yoshihiro Ito

Graduate School of Material Science, NAIST
8916-5 Takayama-cho, Ikoma, 630-01, JAPAN
telephone: +81-743-72-5903, fax: +81-743-72-5903
yito@ms.aist-nara.ac.jp

This is a draft paper for a talk at the
Fifth Foresight Conference on Molecular Nanotechnology.
The final version has been submitted
for publication in the special Conference issue of
Nanotechnology.

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Abstract

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.

Introduction

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

Materials.

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. (Akita, Japan).

Preparation of photoreactive polyallylamine and photoreactive EGF.

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. 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 ether.

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 microscope.

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.

Results

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 immobilized EGF.

Cell growth

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.

Discussion

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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